Introduction

Highly efficient nutrient recycling is essential in coral reef environments, and studies have shown that dissolved organic phosphorus may be more effectively used than dissolved organic nitrogen in reef environments (Padayao and San Diego-McGlone, 2000). Phosphonates, which contain a carbon–phosphorus bond, can account for 25% of the dissolved organic phosphorus pool in marine environments (Dyhrman et al., 2006) and for 10% of cellular particulate phosphate in Trichodesmium (Dyhrman et al., 2009), and are important in marine microbial populations (Benitez-Nelson et al., 2004; Karl et al., 2008; Gilbert et al., 2009; Ilikchyan et al., 2009; Martinez et al., 2009). Phosphonates are, by virtue of the carbon–phosphorus bond, both thermally and hydrolytically stable, and form lipid and protein conjugates widely distributed in the Cnidaria (Hilderbrand, 1983). The cleavage of the carbon–phosphorus bond and subsequent regeneration of phosphate is catalysed by a series of substrate-specific phosphonohydrolases (Kulakova et al., 1997; Quinn et al., 2007), the genetic transcription of which is not pho regulated, but is rather, substrate dependent. Given the prevalence of the phosphonate moiety in coral membrane conjugates, we postulated the existence of microbial phosphonohydrolase-catalysed phosphonate turnover. Previous studies have shown the presence of phnA in open ocean samples (Gilbert et al., 2009). This study further describes the increased diversity of phnA in coral-associated bacteria.

Coral-associated bacteria contain phnA gene homologues

To search for the presence of phnA gene homologues, DNA was extracted from the holobiont of 13 tropical and temperate corals obtained from the National Marine Aquarium, and then PCR amplified using the degenerate nucleotide primers and protocol described by Gilbert et al. (2009). phnA homologues were detected in eight of the holobionts tested (Table 1). The presence of phosphonolipds in these eight corals was confirmed using the lipid extraction and thin layer chromatography method described by Stillway and Harmon (1980), resulting in characteristic blue spots on the thin layer chromatography plate that did not fade over time.

Table 1 Presence (+) and absence (−) of the phnA gene in bacteria associated with various tropical and temperate coral holobionts
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Coral-associated bacteria contain distinct and more diverse communities of phnA gene homologues than previously described in open ocean environments

Clone libraries were prepared from the phnA PCR products obtained from two of these species, Sinularia sp. and Discosoma sp., using the methods described in the study by Gilbert et al. (2009). A total of 256 phnA sequences were characterized (Supplementary Figure S1). As can be seen from Table 2, phnA gene homologues found in both Sinularia and Discosoma are more diverse than those previously described in the open ocean, with clones forming 13 groups with 97% sequence homology in this study as compared with 5 groups from the Western English Channel (Gilbert et al., 2009).

Table 2 Summary of diversity indices derived from phnA gene homologues obtained from the corals Sinularia sp, Discosoma sp and the previous study by Gilbert et al. (2009)
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PhnA gene homologues cluster in groups that are both coral and species specific

As shown in Figure 1, when unique phnA sequences are clustered at 63% sequence identity (the lowest sequence identifies which defines non-overlapping groups), there are three coral-specific and two coastal water-specific groups (the latter obtained from the study by Gilbert et al., 2009), as well as two mixed groups. There is also a Discosoma-specific group that suggests that there are phnA sequence types, which are found only in specific holobionts.

Figure 1
figure 1

Nonmetric multidimensional scaling plot of the unique phnA sequences from coral species, coral-associated bacterial isolates and coastal pelagic samples (Gilbert et al., 2009). All sequences-identity comparisons (Clustal W) were used to create a sequence identity matrix. ▪—coastal water-derived sequences, —Sinularia-derived sequences, —Discosoma-derived sequences, —group 2-cultured isolates, ▴—group 1-cultured isolates. 2D Stress: 0.09.

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PhnA gene homologues from isolates cultured from the coral tissue largely cluster in one group, despite the distant relationship of the host bacteria

To identify the bacterial species that contained these phnA homologues, samples of Sinularia spp. coral tissue were plated onto a marine agar containing phosphonoacetate as the sole carbon and phosphorus source (Gilbert et al., 2009). Isolate taxonomy was verified by 16S rDNA sequencing as described in the study by Gilbert et al. (2009), resulting in 16 distinct bacterial taxa (<97% nucleotide identity). The phnA status of representatives of each taxon was determined using the protocol described in the study by Gilbert et al. (2009). Only eight of these taxa had a phnA homologue, five Gammaproteobacteria (Vibrio, Pseudoalteromonas, Alteromonas, Psychrobacter), one Alphaproteobacteria (Thalassospira), one Actinobacteria (Mycobacterium) and one Bacteroidetes (Flavobacterium). However, the homology of phnA genes was not reflected in the phylogeny of the host bacteria (Supplementary Figure S1). For instance, seven of the eight sequences cluster as a near identical group (group 1); only the Alteromonas phnA sequence does not (group 2; Figure 1). This may reflect the highly conserved nature of the active site of the phnA gene or possibly infer lateral gene transfer between taxa. The eight phnA isolates indicate either the presence of another pathway or a different class of phosphonoacetate hydrolase genes not amenable to amplification with these primers.

Potential coral pathogens contain multiple phosphonate degradation pathways

In all, 3 strains (97% 16S rDNA nucleotide identity) of the 50 cultured isolates belonged to the genus Vibrio (which includes several proposed coral pathogens (Reshef et al., 2006). All sequenced Vibrio representatives in the NCBI database contain the phosphonatase pathway encoded by phnW (2-AEP transaminase) and phnX genes (phosphonoacetaldehyde hydrolase). phnX was identified in all Vibrio isolates using degenerate primers designed to amplify a 154-bp fragment of the phnX gene (PA154R CAATSACRTTYTTSAGTGCC; PA154F ATCGGNCTTGYTCTGGTTA). It is believed the phosphonoacetaldehyde from the transaminase reaction is also converted into phosphonoacetate by an unidentified enzyme; hence, phnA could have a role in this phosphonatase pathway (Quinn et al., 2007).

This study constitutes the first direct identification of phnA homologues from a coral holobiont. The phnA pathway is prevalent within both commensal bacteria and potential coral pathogens, and has a greater diversity than previously found in coastal pelagic waters. Work to determine whether the presence and expression of this functional gene in Cnidarian-associated bacteria is important in both the recycling of phosphorus within reef systems, and the potential pathogenicity of microorganisms associated with coral disease is ongoing.