Study of phylogenetic consistency, structure and origin of the pink berries in Great and Little Sippewissett salt marsh Miniproject Report, Microbial Diversity Course, MBL, 2011 Verena Salman Abstract In the past 20 years pink microbial aggregates from the Little and Great Sippewissett salt marsh have been studied in the microbial diversity course, Woods Hole, Massachusetts. Clone libaries revealed that the aggregates may be dominated by two main phylotypes 1) Halochromatium sp., belonging to the purple sulfur bacteria of the Gammaproteobacteria, and 2) Desulfofustis sp., characterized as a sulfate-reducing bacterium in the Deltaproteobacteria. Furthermore, the biomass of the aggregates was found to be dominated by two morphotypes: 1) large, spherical and pigmented cells and 2) smaller, rod-shaped bacteria. The physical arrangement and their phylogenetic relationship to the Gamma- and Delatproteobacteria, respectively, have been demonstrated. In this study, cluster-specific FISH-probes for the Halochromatium-related and the Desulfofustis-related phylotypes of the berries were designed to serve the confirmation that these phylotypes are forming the main biomass of the berry aggregate. Furthermore, they were used for the specific description of cell arrangement within the aggregate and were tested on water and sand samples in order to reveal the putative presence of free-living stages of the phylotypes. By this, putative early stages of aggregate formation were found in pink sand banks, giving insights into the potential origin of the berries. Isolation of the putative sulfate- reducing bacteria from the berries should provide a tool for studying the metabolites used and products excreted by these organisms, which could help to understand the physiological interconnection of the two dominating organisms in the aggregate. Introduction Large, macroscopic microbial aggregates, so-called ‘pink berries’ from Little and Great Sippewissett salt marsh, Massachusetts, have been studied in the microbial diversity course at MBL, Woods Hole, since more than twenty years. Clone libraries of the 16S rRNA genes have been generated (Banin, 1997; Scott, 2007; Wilbanks et al., 2010) in order to identify the phylotypes present in the aggregates. Two main phylotypes have been identified: 1) Halochromatium sp., belonging to the purple sulfur bacteria of the Gammaproteobacteria, and 2) Desulfofustis sp., characterized as a sulfate-reducing bacterium in the Deltaproteobacteria. The third most abundant phylotype was Cytophaga sp., which are heterotrophic, aerobic bacteria of the Bacteroidetes class. In a previous study (Wilbanks et al., 2010), specific fluorescence in situ hybidization-probes for Deltaproteobacteria and Cytophaga spp. demonstrated the dominance of these phylotypes in the berries. The main biomass of the berries, however, is composed of bacteria that feature a large (3 µm), spherical morphology, that contain red pigments and that are considered to represent the Halochromatium- related phylotype. Until today, however, it has never been demonstrated that the sequences forming the two main phylogenetic clusters in clone libraries (Halochromatium-related and Desulfofustis-related) originate from organisms forming the main biomass in the macroscopic aggregates. Assuming that these two phylotypes are also the physiological keyplayers of the berry consortium, their physical association might play a functional role. Wilbanks et al. (2010) suggested an internal sulfur cycle between the Halochromatium-related and Desulfofustis-related phylotypes. NanoSIMS measurements revealed the incorporation of 34S into the large, round morphotypes, which were thus identified as the purple sulfur bacteria based on morphology, after 34 2- incubation of aggregates with SO4 . It was speculated that the other phylotype, which is assumed to be represented by the smaller, rod-shaped morphotype, performs sulfate reduction, because of their phylogenetic relation to sulfate-reducing Desulfofustis/Desulfocapsa species. Sulfate-reduction results in the formation of sulfide, which may fuel anoxygenic photosynthesis of the purple sulfur bacteria in the Sippewissett aggregates. A physical association of these two partners (large purple sulfur bacteria and small putative sulfate reducer) realizing this internal sulfur cycling was assumed and indicated by Wilbanks et al. (2010), who localized the putative sulfate-reducer with a Deltaproteobacteria-specific FISH probe and found them predominantly attached to large cell clusters of purple sulfur bacteria. They described the arrangement of the large, spherical purple sulfur bacteria (detected by their autofluorescence) and the smaller, rod-shaped Deltaproteobacteria as patchy islands, surrounded by an extrapolymeric substance, containing many Cytophaga filaments (Wilbanks et al. 2010). Large berry-aggregates found exclusively in saltwater pools in the Sippewissett marsh, however, the origin and formation of the aggregates was suggested to be in the sand banks surrounding the water pools and spartina areas. In the sand banks, single sulfur bacteria were detected that resemble the morphology of the main morphotype found in the pink aggregates and that were shown to spontaneously aggregate when shaken for a few seconds in seawater (Seitz et al. 1993). Material and methods Sampling Aggregates were sampled from two pools in Little Sippewissett and one pool in Great Sippewissett. Pool water was collected from the first large pool in Little Sippewissett and pink sand was recovered from top layer of the tide-ways also in Little Sippewissett. Community analysis by 16S rRNA gene pyrosequencing Berries from two pools in Little Sippewissett and one pool in Great Sippewissett were washed three times sterile-filtered marsh water. As berries were bigger in Little Sippewissett, 6 (dataset V7) and 8 (dataset V8) berries were picked from two pools from this site and 15 of the smaller berries were picked from Great Sippewissett (dataset V6). The aggregates were crushed with a pestle (blue pipette tip closed at the front with the Bunsen burner), and were three times frozen at -20°C and thawed at room temperature. The homogenized berries were pipetted into the bead tube of the MoBio power soil DNA extraction kit (CA, USA), 60 µL of buffer C1 was added and the tube was bead-beaten for 30 seconds. The samples were then incubated at 63°C for 5 minutes before the DNA was extracted according to manufactures instructions. Probe design The probe design tool of the ARB software package (Ludwig et al., 2004) was used to design specific probes for the two main phylogenetic clusters found in previous clone libraries from the aggregates (Scott, 2007; Wilbanks et al., 2010). For the purple sulfur bacteria-phylotype, 71 full-length 16S rRNA gene sequences were selected and the probe sequence suggested by the program matched 69 sequences of these. Specificity for the target sequence cluster was tested with the SILVA database (release 102, Pruesse et al. 2007), the RDP and ncbi database, and no hits for sequences outside of the target cluster could be detected. The probe name is PSB-PiBe461 and its sequence is 5´-acgcccaagggtattaac-3´. A mismatch-clone to this sequence was available (Thiodictyon bacillosum or T. elegans), but was not tested in this study (single mismatch three nucleotides in from the 3´end). For the Desulfofustis-related phylotype, 106 full-length 16S rRNA gene sequences were selected and the suggested probe sequence matched 52 sequences of these. Specificity for this sequence cluster was likewise tested with the SILVA database (release 102, Pruesse et al. 2007), the RDP and ncbi database, and also no hits for sequences outside of the target cluster could be detected. The probe name is SRB-PiBe213 and its sequence is 5´-tcctcctcgcacaaccgc-3´. A mismatch-clone to this sequence was not available. Newly designed probes were ordered as horseradish-peroxidase-conjugate from biomers (Ulm, Germany) and diluted to 50 ng/ µL with DNA-grade water. Embedding and cryo-sectioning of aggregates Berries from Little and Great Sippewissett were washed three times in filter- sterilized marsh water and placed into PBS for 1 minute. The PBS was removed with a pasteur pipette and a solution of 4% formaldehyde and 0.5% glutaraldehyde (in MilliQ) was added. The aggregates were fixed for 1 hour at room temperature and the liquid was removed again with a pasteur pipette. Fresh PBS was added and removed three times to wash the berries before a 7.5% sucrose solution (in PBS) was added. The cells were incubated for 1 hour while slowly rotating at room temperature. Sucrose-solution was removed with a pasteur pipette, PBS was added and removed again. O.C.T. Tissue Tek (Sakura, CA, USA) was added, well covering the aggregates. They were rotated for 16 hours at room temperature and then incubated standing at 4°C for 24 hours. Single aggregates were moved into cryo-freezing molds, which were then placed horizontally into liquid nitrogen for several minutes. The molds were kept at -80°C for 12 hours before placed at -20°C for two days. Embedded aggregates were sectioned with a cryo-microtom HM 505 N into 20 µm sections and placed onto polysine-covered glass slides (Thermo Fisher Scientific Inc., Schwerte, Germany). Slides were stored at -20°C until used. Fixation and filtration of pool water and pink sand Pool water (36 mL) from Little and Great Sippewissett was mixed with 4 mL of 20% formaldehyde (4% final concentration) and incubated for 40 minutes at room temperature and for 3 hours at 4°C. A support-filter (0.45 µm pore size) was placed onto a filtration device before the 0.2 µm membrane filter was placed on top. Fixed pool water was filtered and filter tower was washed by filtering 20 mL fresh PBS. Filter was placed on whatman paper to dry and stored at -20°C. Pink sand from sand banks (0.5 mL) from Little and Great Sippewissett was mixed with 0.5 mL of 4% formaldehyde (in PBS, 2% final concentration) and incubated for 40 minutes at room temperature. The sample was centrifuged at 10,000 xg for 10 minutes and the supernatant was discarded. The sand was resuspended with PBS by inverting the tube and centrifuged again.