Natural Communities of Novel Archaea and Bacteria with a String-of-Pearls-Like Morphology: Molecular Analysis of the Bacterial Partners

Abstract

A recently discovered bacterial/archaeal association, growing in a string-of-pearls-like structure, thrives in the cold (∼10°C) sulfidic marsh water of the Sippenauer Moor near Regensburg, Bavaria, Germany. It forms characteristic, macroscopically visible globules, the pearls, containing microcolonies of novel euryarchaeota, which are surrounded by mainly filamentous bacteria (C. Rudolph, G. Wanner, and R. Huber, Appl. Environ. Microbiol. 67:2336-2344, 2001). Single pearls in series are connected by white threads. Here we report the first detailed molecular investigations of the taxonomic affiliation of the bacteria contributing to the strings of pearls. Phylogenetic analysis showed the dominance of a single phylotype (clone sipK4) within single pearls most closely related to Thiothrix unzii. The presence of Thiothrix sipK4 as a major constituent of single pearls and of the pearl-connecting white threads was verified by fluorescence in situ hybridization.

Microbial communities growing as biofilms (7, 13, 25, 33, 37), microbial mats (22, 32, 35), and consortia (1, 26) are a widespread phenomenon in nature. Such microbial associations are mostly found in natural aquatic systems, in artificial medical environments, and as epi- or endoliths (6, 13). Living in a community provides many advantages for the constituent microorganisms: environmental changes are buffered, nutrient uptake is easier, and horizontal gene transfer is possible (9, 23, 28). Sulfide-oxidizing, filamentous bacteria like Beggiatoa, Thioploca, and Thiomargarita species frequently form an integral part of microbial mats (11, 20, 29); Thiothrix is often associated with activated sludge bulking (10, 34), but is also found in mats in natural aquatic environments (21).

In the last few years, molecular methods have been developed that allow the study of complex microbial associations without cultivation. DNA isolation, PCR, cloning, and sequencing are used to obtain clone libraries of 16S rRNA gene sequences from naturally occurring microbial communities (see references 31 and 35 and references therein). Fluorescence in situ hybridization (FISH) with 16S rRNA probes is another molecular approach to determine the in situ composition of microbial communities (2, 24). Very recently, FISH studies showed the abundance of a marine microbial consortium, comprising archaea of the order Methanosarcinales and sulfate-reducing bacteria of the δ-Proteobacteria (4). These microbial assemblages appear to mediate anaerobic oxidation of methane in gas-hydrate-rich sediments. A combination of 16S rRNA clone libraries and FISH studies may, therefore, be the best method to characterize the in situ composition of microbial communities by using adapted genus-specific or species-specific fluorescent probes (35, 36).

Recently, a naturally occurring association of a novel group of archaea and bacteria was discovered in cold (∼10°C), sulfide-rich marsh water of the Sippenauer Moor near Regensburg, Bavaria, Germany (27). This macroscopically visible, whitish-colored community grows in a string-of-pearls-like structure (Fig. 1). Tiny, whitish pearls (diameter, 0.5 to 3.0 mm) are connected to each other by thin, white threads. FISH studies showed that, in the inner part of smaller single pearls, archaeal cocci predominate, growing as microcolonies (27). The archaea (estimated at up to 10⁷ cells per pearl) appear to be embedded in a polymer of so far unknown chemical composition. Detailed investigations of periodically collected pearls have shown that these archaea belong to a single euryarchaeal phylotype, representing a deep phylogenetic branch within the 16S rRNA tree. The outer part of the pearls is mainly composed of bacteria with a predominantly filamentous morphotype (27). To get a better insight into the microbial diversity, composition, and architecture of single pearls and the pearl-connecting threads, the bacterial counterparts of the archaea have been studied in more detail by a combination of 16S ribosomal DNA (rDNA) sequence analysis and FISH.

FIG. 1.

Photograph of archaeal and bacterial communities growing in a unique, string-of-pearls-like morphology in the sulfurous water of the Sippenauer Moor. Arrows point to single pearls. Bar, 3 cm.

Collection and preparation of samples.

Samples were collected from the sulfurous water of the main spring of the Sippenauer Moor near Regensburg, Bavaria, Germany (27). Before sampling, the sampling equipment was treated with 5% HClO₄ to get rid of DNA impurities. Pearls for DNA extraction were kept cold (at 4°C) in a refrigerated box; single pearls and connecting threads for FISH were immediately fixed by adding formaldehyde (final concentration, 3% [wt/vol]). All pearls sampled had a diameter between about 0.5 and 3 mm.

DNA isolation, PCR, cloning, RFLP, sequencing, and phylogenetic analyses.

Cell lysis and bulk DNA extraction were performed as described previously (27). Extracted DNA was used as template for the PCR amplification of bacterial 16S rDNA sequences with the bacterium-specific forward primer 9f (5) and the universal reverse primer 1392r (14). PCR was performed as described earlier (27). The PCR product was purified following electrophoresis on a 1% (wt/vol) low-melting-point agarose gel for 90 min at 70 V. The ethidium bromide-stained DNA band was visualized under UV light and excised. DNA in the agarose slice was recovered with GeneClean II (Bio101, Inc., Vista, Calif.) according to the manufacturer's instructions. Cloning of the PCR products, restriction fragment length polymorphism (RFLP), and sequencing were carried out as described previously (27). Clones with unique RFLP patterns were chosen for sequencing. The sequences were amplified with the bacterial primer 9f (5) and the universal primers 516uF (sequence [5"→3"], TGB CAG CMG CCG CGG TAA) (K. O. Stetter and S. Burggraf, unpublished data) and 1392r (14). Phylogenetic analyses were done as previously described (27).

Oligonucleotide probes, FISH, and DAPI stain.

For FISH, the pearls were gently flattened with pipette tips. Whole-cell hybridization was carried out by the use of the Archaea-specific Arch915 probe (rhodamine green labeled) (30) and the Thiothrix-specific TN1 probe (CY3 labeled) (34) on a single spot. FISH and epifluorescence microscopy were performed as recently described (27). The hybridization solution and the washing buffer for FISH contained 0.01% sodium dodecyl sulfate. After the FISH procedure, each sample was stained with 10 μl of 4",6"-diamidino-2-phenylindole (DAPI; 2 mg/liter; prepared in washing buffer).

Classification of the bacterial clone sequences.

RFLP analysis of 134 bacterial clones resulted in 19 different bacterial 16S rDNA sequences. None of the sequences was judged to be chimeric. Phylogenetic classification showed that the sequences were spread over several bacterial phyla. The distribution of the 134 bacterial clone sequences was as follows: green nonsulfur, 1.5%; Clostridium and relatives, 2.2%; β-Proteobacteria, 3.0%; high-G+C gram positives, 3.0%; ɛ-Proteobacteria, 4.5%; δ-Proteobacteria, 5.2%; α-Proteobacteria, 5.2%; Planctomycetales, 6.0%; Bacteroides-Cytophaga-Flexibacter group/green sulfur bacteria, 8.2%; and γ-Proteobacteria, 61.2%. The phylogenetic position of all clone sequences was verified by different tree reconstruction methods. The maximum parsimony bacterial phylogenetic tree is shown in Fig. 2. One sequence predominated out of all the sequences derived. About 60% belonged to a single phylotype (clone sipK4) within the γ-Proteobacteria, with the closest relationship being to Thiothrix unzii (Fig. 2) (12). Phylogenetic analysis of another pearl studied 1 year earlier showed a similar dominance of the identical Thiothrix sequence.

FIG. 2.

16S rRNA gene-based phylogenetic tree showing the position of the bacterial sequence clones derived from a pearl. The topology of the tree is based on results of a maximum-parsimony analysis (ARB software package) (19). Reference sequences were chosen to represent the broadest diversity of Bacteria (scale bar, 10% estimated difference in nucleotide sequence positions). The accession numbers of the sequences occur after the species names and clone designations.

Microscopic studies.

Phase-contrast microscopy of gently squeezed pearls on microscopic slides showed the dominance of rod-shaped, filament-forming microorganisms (Fig. 3). The average diameter of the filaments was 2 μm. Motility of the filaments was not observed. Very often, the filaments formed rosettes (Fig. 3). The filaments contained large amounts of refractile globules. Transmission electron microscopy (TEM) and energy-dispersive X-ray analysis (EDX) studies revealed that the globules consisted of amorphous sulfur. The same sulfur modification was also identified within an isolate of Thiothrix fructosivorans, recently obtained from the Sippenauer Moor (J. Raabe, Institut für Experimentelle und Angewandte Physik, Universität Regensburg, personal communication).

FIG. 3.

Phase-contrast micrograph of the outer part of a pearl, showing filamentous, rosette-forming microorganisms with sulfur globules. Bar, 20 μm.

FISH studies.

In accordance with the phylogenetic studies, the majority of the filamentous bacteria exhibited a positive hybridization signal with a Thiothrix-specific hybridization probe (Fig. 4A). The DAPI stain verified that most of the bacterial cells (estimated up to approximately 95% in close vicinity to the archaeal microcolonies of small single pearls) could be attributed to the genus Thiothrix (Fig. 4B). About 30 single pearls sampled over a period of half a year showed the same characteristic composition of coccoid archaea and Thiothrix sipK4 as the predominant bacterium. FISH studies of the pearl-connecting whitish threads revealed that Thiothrix was again very often the predominant bacterium. Further filamentous and so-far-unidentified bacteria, together with a eucaryotic alga, were also observed. Almost no archaeal cocci were found within the threads. Studies of different threads over a period of several months showed a similar microbial composition.

FIG. 4.

(A) FISH of a part of a pearl. An epifluorescence micrograph, dually hybridized with a rhodamine green-labeled archaeal probe (ARCH915) and a CY3-labeled Thiothrix-specific probe (TN1), is shown. The archaeal cocci stain green, and Thiothrix stains red. (B) DAPI stain of the same sample. Bar, 10 μm.

Conclusions.

Bacterial phylogenetic analysis of a single pearl revealed that most clone sequences (about 60%) belonged to a single phylotype most closely related to Thiothrix unzii. This result indicates that the identified Thiothrix clone sipK4 is a major bacterial constituent of the pearl community. The remaining clone sequences were spread over different bacterial phyla. At the moment, it remains unclear if these sequence-predicted organisms were genuine members of the pearl or were randomly attached. In the future, this could be investigated by periodic screening of pearls with terminal RFLP, which allows the observation of population dynamics within microbial associations (17, 18).

However, it has been reported that clone libraries of 16S rDNA sequences can deviate from the real composition of communities due to biases at each step of the method (2, 8, 39). FISH was therefore chosen to cross-check the phylogenetic analysis. The presence of Thiothrix as a major constituent of a pearl was verified by the use of a genus-specific hybridization probe and DAPI stain (34). The predominance of Thiothrix in different strings of pearls was confirmed by (i) FISH studies of about 30 single pearls, collected over a period of about 1 year; (ii) a second, independent phylogenetic study of a single pearl; and (iii) microscopical investigations of pearls and threads.

Thiothrix itself is a typical aerobic, sulfide-oxidizing bacterium of freshwater habitats (3, 12, 15, 38) and, therefore, is well adapted to its natural biotope, Sippenauer Moor. Different functions of Thiothrix clone sipK4 within the string-of-pearls-like community can be envisaged. The most obvious role can be seen in the formation and maintenance of the three-dimensional structure of the outer part of the pearls and the pearl-connecting white threads. A characteristic feature of members of Thiothrix is the ability to attach to different solid surfaces by using its gonidial holdfast material (16). Therefore, Thiothrix sipK4 could also play an important role in primary settlement, potentially the first step in the formation of the string-of-pearl community.

The coexistence of the novel archaeal group and Thiothrix sipK4 was observed over a longer period by the study of different pearls. Therefore, a syntrophic or even symbiotic relationship between these two groups can be imagined (28). Members of the genus Thiothrix are known to grow by aerobic sulfide oxidation, forming sulfate as an end product. An anaerobic environment could be created for the archaeal cells, and sulfate could be supplied as a possible electron acceptor for growth. The sulfide produced by the archaea might be used by Thiothrix, creating a sulfur cycle within a pearl. Furthermore, different metabolic products formed during growth by Thiothrix might be suitable carbon and energy sources for the archaeal partner (12). The string-of-pearls-like communities in the Sippenauer Moor therefore appear to represent extraordinary and stable associations of defined groups of microorganisms belonging to different domains of life.

Nucleotide sequence accession numbers.

The 16S rRNA gene sequences of all derived bacterial clones were deposited in the EMBL Nucleotide Sequence Database: accession numbers are given in Fig. 2.