Novel Epibiotic Thiothrix Bacterium on a Marine Amphipod

Comparative analysis of the 16S rRNA gene and fluorescent insitu hybridization (FISH) was used to identify epibiotic filamentousbacteria living on the marine amphipod crustacean Urothoe poseidonis.The epibionts belong to the gamma proteobacteria and representa novel marine phylotype within the genus Thiothrix. FISH anddenaturing gradient gel electrophoresis revealed that the Thiothrixfilaments are present on the majority of the amphipods examined.

INTRODUCTION

Top

Introduction

Bacteria of the genus Thiothrix are filamentous sulfur-oxidizingorganisms that produce gliding gonidia, form holdfast structurescalled rosettes, and deposit intracellular sulfur granules whengrown in the presence of sulfide or thiosulfate (12, 18). Thiothrixspp. have been described from a number of habitats, rangingfrom sulfide-containing natural waters and irrigation systems(3, 4, 18) to activated-sludge wastewater treatment plants,where they contribute to the problem of filamentous sludge bulking(15, 21).

Some authors have suggested that Thiothrix spp. can also occuras epibionts on aquatic invertebrates such as mayfly larvae(17), tadpoles (7), hydrothermal vent organisms (14), intertidalsediment dwellers (9, 22), and the sea urchin Echinocardiumcordatum (2, 26). However, these Thiothrix-like bacteria weremainly identified on the basis of their morphology (2, 7, 9,17) or by using immunological methods (2). It is now known thatmorphology alone is of little use in identifying Thiothrix spp.because supposed defining features such as rosettes and gonidiaare also found in other filamentous bacteria (12). To illustratethat morphology can be misleading, a recent report demonstratedthat the filamentous bacteria in the digestive tract of E. cordatum,assumed for years to be Thiothrix-like bacteria (2, 26), arein fact Desulfonema spp. (28).

Comparative sequence analysis of the 16S rRNA gene has revealedthat the true members of the genus Thiothrix form a well-definedmonophyletic group within the gamma subdivision of the Proteobacteria(12, 15, 24, 27). To date, none of the Thiothrix spp. withinthis clade have been isolated from marine habitats or have beendescribed as epibionts of aquatic invertebrates.

Urothoe poseidonis is a small amphipod crustacean (±4mm in length) that lives freely in marine sediments or as acommensal in the burrows of various endofaunal invertebrates(10, 16, 29). Three morphotypes of filamentous bacteria havebeen described as epibionts on the walking appendices (the fifthpair of pereopods) of U. poseidonis, one of which shows thetypical Thiothrix morphology (10). To date, these Thiothrix-likebacteria could not be cultivated using the various media describedearlier for Thiothrix spp. (30). The aim of the present workis to identify these Thiothrix-like bacteria living on U. poseidonisby using molecular methods and in situ hybridization.

16S rRNA cloning and phylogenetic analysis.

Top

16S rRNA cloning and...

Individuals of U. poseidonis were collected from burrows ofthe sea urchin E. cordatum at Wimereux, Pas de Calais, France,in January of 2002. The bacteria from several amphipods werepooled and DNA was extracted using the freeze-thaw method (11).The PCR products, amplified with the bacterial primers 8F and1492R for the 16S rRNA gene (5), were cloned into the pCR4-TOPOvector (Invitrogen). Thirty-seven clones were partially sequencedwith the bacterial primer 518F (5) on an ABI Prism 3100 geneticanalyzer and submitted to BLAST (20). Only one partial sequencegrouped with known Thiothrix species, clone UP23b. This clonewas sequenced completely (1,464 bp) by using the described bacterialprimers for the 16S rRNA gene (5). The complete UP23b sequencewas then aligned manually to the 16S rRNA sequences of closeevolutionary relatives by using the sequence alignment editorSeqPup v0.6f (8). Phylip v.3.6a3 (6) was used to estimate maximum-likelihoodtrees on the basis of a data matrix of 1,265 characters (weused empirical base frequencies, a Ti/Tv ratio of 2, one categoryof unweighted sites, and a constant rate of variation amongsites). Parsimony and maximum-likelihood bootstrap analyseswere performed with 100 replicates using Phylip and the ARBsoftware package (19). As shown in Fig. 1, clone UP23b groupedwith other known Thiothrix species with bootstrap values of99% (parsimony) and 77% (maximum likelihood). The highest similarityvalue observed was between clone UP23b and Thiothrix eikelboomiiAB042819 (93.2%). The 16S rRNA sequence signature of the genusThiothrix, a characteristic deletion in the stem-loop structurecorresponding to positions 455 to 477 (Escherichia coli numbering),was found in clone UP23b (12).

View larger version (30K):
[in this window]
[in a new window]
FIG. 1. Phylogenetic relationship of the U. poseidonis clone 23b based on comparative 16S rRNA sequence analysis using maximum likelihood (matrix of 1,265 nucleotides). Branch lengths do not represent the true distances in the consensus tree. Members of the gamma subdivision of the Proteobacteria are shown and GenBank accession numbers are listed for all sequences. Bootstrap values above 50% (obtained with 100 resamplings) are shown, with upper and lower values representing those from parsimony and maximum likelihood, respectively. E. coli served as the outgroup.

Probe design and fluorescent in situ hybridization (FISH).

Top

Probe design and fluorescent...

Oligonucleotide probes were constructed using the PROBE_DESIGNtool of the ARB software package (13, 19). The designed probe(UP23b: 5'-CTCGGCATCCTGTCCACG-3') targeted positions 1033 to1050 (E. coli numbering) of the 16S rRNA molecule and had atleast one central mismatch to all other known 16S rRNA sequencesas determined by Check Probe 2.1r from the Ribosomal DatabaseProject II (http://rdp.cme.msu.edu/). The probe was labeledat the 5' end with the indocarbocyanin dye CY3. For FISH, wholeanimals were fixed in 4% paraformaldehyde, rinsed, and keptin ethanol-sterile seawater (50:50) at –20°C. Pereopodswith the attached bacteria were then separated, placed in 500µl of ethanol-sterile seawater, and sonicated briefly.Five microliters of the suspension were deposited on gelatin-coatedslides, hybridized at 46°C, and viewed as described elsewhere(23). Hybridizations were performed under stringent conditionswith 60% formamide in the hybridization buffer. The probes EUB338,NON338, and GAM42a were used as positive and negative controls(1). In situ hybridization with probe UP23b yielded a strongfluorescent signal (Fig. 2). The labeled filaments were composedof disk-like cells of about 3 µm in diameter and about1 µm in length. Thiothrix filaments were detected on 7out of 10 amphipods examined. The Thiothrix filaments, whichwere also labeled with the EUB338 and GAM42a probes, were neverabundant on individual amphipods (0 to 5 filaments of about25 to 300 µm in length on each pereopod no. 5). Althoughother morphotypes of epibiotic bacteria are known to occur onU. poseidonis (10), only the Thiothrix-like filaments hybridizedto the UP23b probe. Epibiotic filamentous bacteria have alsobeen described from the shells of the bivalve Montacuta ferruginosa,another invertebrate living in the burrow of E. cordatum (9,11), and have been reported to occur in the nodules of the digestivetube of E. cordatum (2, 26). These filamentous bacteria didnot hybridize with the UP23b probe. Furthermore, Thiothrix-likefilaments were never found in the sediments of the E. cordatumburrow. These results indicate that at least at the site investigatedin this study, the Thiothrix phylotype is only associated withU. poseidonis and does not occur in or on other invertebratesor free-living in the sediment.

View larger version (99K):
[in this window]
[in a new window]
FIG. 2. FISH of the Thiothrix-like bacteria from U. poseidonis with the specific probe UP23b. The filamentous bacteria are attached to setae (A and B) or to spines (C and D) of the amphipod. Identical microscopic fields were visualized with an epifluorescence microscope using filter sets specific for DAPI (4',6'-diamidino-2-phenylindole) (A and C) and the CY3 fluorochrome (B and D). Scale bars, 10 µm.

Denaturing gradient gel electrophoresis (DGGE).

Top

Denaturing gradient gel...

DNA from 10 individuals (5 males, 5 females) was extracted asdescribed above. PCR products, amplified with the bacterialprimers GM5F-GC-clamp and 518R (11), were separated on 25 to75% gradient gels (11). DGGE analysis revealed a complex epibioticmicrobial community. Of the 26 band positions detected withDGGE, only 5 bands were present in all 10 DGGE profiles (Fig.3, bands a to e). These could represent common epibiotic bacteriabut could also represent seawater bacteria. Band e migratedto the same position as clone UP23b. To check the identity ofband e, it was excised from three DGGE profiles and reamplifiedusing primer GM5F without a GC clamp (11). The sequences ofband e were 100% identical to the sequence of clone UP23b (±150bp of the DGGE fragments could be sequenced). This indicatesthat the Thiothrix phylotype was present on all 10 amphipodsexamined.

View larger version (93K):
[in this window]
[in a new window]
FIG. 3. DGGE of PCR-amplified 16S rDNA fragments from the epibiotic microbial communities of 10 amphipods (lanes 1 to 10). The same 16S rDNA fragment was amplified by PCR for clone UP23b (lane 11). The five DGGE bands that are present in every profile are indicated on the left (a to e).

Concluding remarks.

Top

Concluding remarks.

This study demonstrates for the first time that a Thiothrixspecies thrives in the marine environment as an epibiont ofan amphipod. These bacteria have the characteristic Thiothrix16S rRNA sequence signature and the key morphological featuresof Thiothrix spp. As the rRNA sequence of clone UP23b differsfrom other Thiothrix species by more than 2.5%, clone UP23bprobably represents a novel Thiothrix species (12, 25). Theability of this new Thiothrix species to oxidize sulfur compoundsand its role in the microbial community associated with U. poseidonisremains to be shown.

Nucleotide sequence accession number.

Top

Nucleotide sequence accession...

The 16S rRNA gene sequence UP23b has been deposited in the GenBankdatabase under accession no. AY426613.

ACKNOWLEDGMENTS

D.C.G. is a senior research assistant of the NSFR (Belgium).We acknowledge the contribution of the Centre Interuniversitairede Biologie Marine (CIBIM).

FOOTNOTES

* Corresponding author. Mailing address: Marine Biology Laboratory, CP160/15, Université Libre de Bruxelles, 50 av. Roosevelt, B-1050 Brussels, Belgium. Phone: 32-2-6502970. Fax: 32-2-6502796. E-mail: dgillan{at}ulb.ac.be.

REFERENCES

Top