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SI Materials and Methods

The whole animals were subjected to genomic DNA extraction using the DNeasy Tissue Kit (Qiagen, Hilden, Germany). Whole animals were lysed in 180 ml of DNeasy ATL buffer and 20 ml of proteinase K for 3 h at 56°C. Two hundred microliters of AL buffer was added to the lysate and incubated for 10 min at 70°C. After the addition of 200 ml of 100% ethanol, the lysates were purified over a DNeasy column and eluted in 100 ml of AE buffer. From the gDNA, bacterial 16S RNA genes were amplified by PCR with the primers 27F (5'-TG(A/G)GTTTGATC(A/C)TGGCT(C/T)AG-3') and 1492R (5'-TGG(A/C/T)TACCTTGTTACGACTT-3') (1). PCR was conducted with 2.5 units of Taq-DNA Polymerase (GE Healthcare, Piscataway, NJ) and its supplemented buffer system, 1 mM each primer, 0.1 mM each dNTP, and 1 ml of extracted DNA in a final volume of 50 ml under a temperature profile of 94°C for 3 min followed by 30 cycles of 94°C for 30s, 53°C for 30s, and 72°C for 1 min 40s. Resulting PCR fragments were cloned into pGEMT vector (Promega, Madison, WI) and transformed into electrocompetent DH5a Escherichia coli cells (Invitrogen, Karlsruhe, Germany). The plasmid inserts were checked by PCR with the vector-specific primers SP6 (5'-ATT TAG GTG ACA CTA TAG AAT AC-3') and T7 (5'-TAA TAC GAC TCA CTA TAG GG-3') for the correct product size (»1,600 bp). The amplified inserts of the 16S rRNA genes were subjected to restriction fragment length polymorphism by using the restriction enzymes HaeIII and Hin6I (Fermentas, Glen Burnie, MD). Representative plasmids were sequenced using a LI-COR 4300 DNA analyzer plate sequencer (LI-COR Biosciences, Lincoln, NE).

Sequences were sorted into phylotypes using the criterion of 97% sequence identity. All of the sequences were subjected to the Check chimera program Bellerophon (2) and RDP II Chimera Check (3) for the elimination of chimeric sequences. Chloroplast sequences were identified and removed. The final dataset of 36 sequences was aligned using the ARB software package (4). Closely related sequences were found by the function "search for the closest relatives" implemented in the ARB software and by a BLAST search and added also to the alignment. Alignments were optimized by hand, and a neighbor-joining tree was calculated with all 16S rDNA sequences and their closest relatives by using Olsen correction.

Macerates were performed according to the standard protocol (5). Hybridizations of fixed mazerated Hydra cells (immobilized on glass slides) were done as described by Manz et al. (6) with monofluorescently labeled rRNA targeted oligonucleotide probes: EUB338, 5'-GCTGCCTCCCGTAGGAGT-3' (universal eubacterial probe, positive control) and nonEUB338 5'-ACTCCTACGGGAGGCAGC-3' (EUB338 antisense probe, negative control). Probes were 5'-end-labeled with either fluorescein (green fluorescence) or Cy3 (red fluorescence). Hybridization were carried out by 46°C, 90 min in followed by one wash step at 48°C, 15 min. Additionally, samples were stained with Hoechst and mounted with Citifluor (Citifluor Ltd., London, U.K.). Examination was done by a ´1,000 magnification with a Zeiss Axioskop 2 epifluorescence microscope by using filter sets for DAPI, Cy3, and fluorescein.

The phylotype-specific oligonucleotide probe HoSym1030 (Cy3, 5-CCTGTGATAGTCCAGCCG-3; E. coli positions 1030-1048) was designed by using the probe design function of the ARB software (4). This probe matched exactly the target region of the 16S rRNA molecule of the dominant phylotype identified in Hydra oligactis (lab). It had one or more mismatches with the sequences of all other bacteria in the Ribosomal Database Project database (7). The formamide concentration in the hybridization buffer was varied between 0 and 30%, while the sodium chloride concentration in the posthybridization buffer was adjusted accordingly. The fluorescence signal by probe HoSym1030 to target cells was stable; the intensity of this signal was stable between 0 and 20% formamide and decreased slightly at 30% formamide. With nontarget cells, there was no signal even under low-stringency conditions (no formamide). Therefore, we routinely used 10% formamide for single hybridizations and for double hybridizations with EUB338.

For transmission electron microscopy, polyps were fixed overnight at 4°C in 3.5% glutaraldehyde in 0.05 M cacodylate buffer (pH 7.4). After removal of glutaraldehyde, samples were postfixed in 0.075 M cacodylate buffer containing 1% OsO₄ for 2 h at 4°C. After washing, dehydration was carried out using ethanol, samples were incubated with 1.2 propylenoxide and embedded in Agar 100 resin (Agar Scientific, Ltd., Essex, U.K.). Ultrathin sections were contrasted using uranyl acetate and lead citrate and analyzed under a transmission electron microscope.

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4. Ludwig W, Strunk O, Westram R, Richter L, Meier H, Yadhukumar, Buchner A, Lai T, Steppi S, Jobb G, et al. (2004) Nucleic Acids Res 32:1363-1371.

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6. Manz W, Amann R, Ludwig W, Wagner M, Schleifer KH (1992) Syst Appl Microbiol 15:593-600.

7. Cole JR, Chai B, Farris RJ, Wang Q, Kulam-Syed-Mohideen AS, McGarrell DM, Bandela AM, Cardenas E, Garrity GM, Tiedje JM (2007) Nucleic Acids Res. 35 (Database issue): D169-D172.