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Characterization of Anoxygenic Phototrophs That Grow Using Infrared Radiation (>800 Nm) (Sampling Location: Little Sippewissett Marsh, Woods Hole, Ma)

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Characterization of Anoxygenic Phototrophs That Grow Using Infrared Radiation (>800 Nm) (Sampling Location: Little Sippewissett Marsh, Woods Hole, Ma) Characterization Of Anoxygenic Phototrophs That Grow Using Infrared Radiation (>800 Nm) (Sampling Location: Little Sippewissett Marsh, Woods Hole, Ma) Martina Cappelletti, PhD University of Bologna, e-mail: [email protected] Microbial Diversity Course Marine Biological Laboratory, June-July 2011 1 ABSTRACT In this study I performed two enrichment cultures for purple bacteria from a mat soil sample collected in Little Sippewissett marsh. I obtained two mixed culture that were named LWS_880 and LWS_960 as they could grow absorbing light at 880 nm and 960 nm. The molecular analysis of the culturable bacteria obtained by using two different isolation techniques was performed with two primer sets, one specific for the 16S rDNA and the other for the pufM gene. As a result, culturable bacteria in LWS_880 were identified as purple non- sulphur bacteria belonging to the following genera Rhodobacter, Rhodospirillum, Rhodovulum and Rhodobium. The culturable bacteria in LW_960 were shown to belong to the genera Thioroducoccus, Allochromatium, Marichromatium of purple sulfur bacteria and to the purple non-sulfur Rhodovolum genus. CARD-FISH hybridization technique allowed measuring the relative abundance of each group of Proteobacteria in each microbial community pointing out interesting differences between the two samples. Further biochemical assay identified the bacterioclorophyll present in each consortium and chemotaxis activity was detected in the sample LWS_960. INTRODUCTION Most of the fossil fuels utilized as energy source on earth is the result of photosynthesis process occurred many hundreds of millions of years ago. Moreover, the evolution of oxygenic photosynthesis resulted in the oxygenation of Earth’s atmosphere creating a radical new environment for all life. These two observations point out how the photosynthesis is an invaluable process to understand at the deepest levels (Bekker et al. 2004). It is likely that some form of anoxygenic photosynthesis was a precursor to the complex machinery necessary for oxygenic photosynthetis (Blankenship, 1992). Because of this, the modern anaerobic phototrophs, belonging exclusively to the bacterial kingdom, represent model systems to study photosynthesis in its simplest forms. The anoxygenic photosynthesis occurs in 4 groups of bacteria: phototrophic green bacteria, phototrophic purple bacteria, Heliobacteria and Acidobacteria. Purple bacteria are divided in purple sulphur bacteria that are able to utilize H2S as electron donor along with other sulphur reduced compounds (as thiosulfate), and purple non-sulphur bacteria that are mainly able to utilize organic compounds as electron donors such as organic, fatty and amino acids, alcohols and aromatic compounds (Overmann, 2001). Anoxygenic phototrophs are taxonomically dispersed among the α-, β- and γ-proteobacteria groups and have bacteriochlorophyll a or b in their photosynthetic reaction center that have 2 absorption maxima in the red and near infrared part of the spectrum (wavelength >700-1100 nm). Due to these peculiar chlorophylls, they can grow in the deeper layers of microbial mats in sandy sediments that are reached only by light in the infrared wavelength range. Indeed, the light in the visible spectrum is almost completely absorbed by cyanobacteria and diatoms composing the upper layers of the mat (Overmann, 2001). In this study I enriched for purple non-sulphur bacteria communities that were able to grow absorbing two different wavelengths of light in the IR region (i.e. 880 nm and 960 nm). The enrichments were performed by using the sediment layer underlying the phototrophic mats in Little Sippewissett marsh. Composition of the anoxygenic phototrophic consortia was assessed by identifying at molecular level the culturable bacteria and by performing CARD- FISH hybridization technique. Biochemical features were also investigated with chemotaxis activity assay and pigments analyses by analyzing the absorption spectra of whole cells samples of the biomass. MATERIALS AND METHODS Enrichment. A microbial mat sample in Little Sippewissett Marsh, Woods Hole, MA was collected in a 50-mL Falcon tubes maintaining the layers stratification existing in the soil. 0.5 gr of the soil laying around 2 cm deep in the sediment was added into “Pfennig bottles” containing 10 mL of Marine Phototrophic Base each. The medium contained the following components: Artificial seawater base, 10 mM NH4Cl, 1 mM KH2PO4, 1 mM NaSO4, 20 mM MOPS buffer, pH 7.2, HCl-dissolved trace elements, Multivitamin solution, 5 mM NaHCO3. 5 enrichments were performed by adding different electron donors and by incubating the cultures at different wavelengths of light, as described in the Table 1. !"#$%&'()*'('& +,-".%/)'0)".12% !(&.$23#(%)0'& !"# @',+*+, $$# 9&:43,5)/)A2&341&:5;*'+,:(*5 %&''()*+, $$# <97%-= -,)./*+, $$# 01(/2&341*+,5*)657*8% $$# 9&:43,52&341&:5;*'+,:(*5<9%-= >8# 7*8% B:,,)52&341&:5;*'+,:(*5<B%-= >?# Table 1. The different conditions of growth including the electron donor and the quality of light used are described. The last column described the type of enrichment expected for each condition. One bottle was set for each condition. Isolation of single colonies in Agar Shake Tubes. 1 mL of each grown enrichment was inoculated into 50 mL glass tube containing 9 mL of anaerobe Marine Phototroph Base 3 supplemented with the appropriate electron donor and 15 g/L of washed agar. Serial diutions were performed in order to obtain single colonies. Shake Tubes were incuated at 30ºC and were illuminated by LEDs emitting the same light that was used to illuminate the liquid enrichment that was inoculated. When the colonies were grown, the agar in the original shake tubes was aseptically blown into sterile petri dishes; the colonies were picked and resuspended in 10 µL of H2O in a sterile 1.5-mL tube for further analyses. Isolates obtained with this cultivation method were named as ST followed by the wavelength of growth. Isolation of single colonies from Agar Plates. 100 µL of each enrichment was spread onto Marine Phototroph Base agar medium containing the appropriate electron donor. The plates were incubated at room temperature anaerobically in GasPak jars in ambient light. After two weeks, the GasPack jars were opened and single colonies were picked up from the plates and resuspended in 10 µL of H20 in a sterile 1.5-mL tube for further analysis. Isolates obtained with this cultivation method were named as PL followed by the wavelength of growth. Phylogenetic analysis of the isolates. 2 µL of each suspension containing a single isolate was used for colony PCRs with two primer sets. The first primer set included the universal bacterial primers 8F and 1492R amplifying the 16S rDNA gene. The second primer set (PB557F-PB750R) targets the pufM gene encoding the M subunit of the photosynthetic reaction center. Since purple sulfur and non-sulfur bacteria are phylogenetically distributed among the α−, β− and γ−proteobacteria (Lee et al., 2005), the 16S rDNA gene may not be an appropriate target for phylogenetic analysis. Recently, Achenbach et al. (2001) developed a functional gene approach to assess the community composition of anoxygenic purple bacteria in natural environments. This approach is based on the molecular analysis of the photochemical reaction centre complex encoded by the puf operon that is universally distributed among purple phototrophic bacteria (Anthony Ranchou-Peyruse et al, 2006). The 16S rDNA sequences obtained were compared with existing sequences in the Ribosomal Database Project. Geneious Pro 4.7.6 software was used to process the nucleotide sequences of pufM gene while homology searches were performed with nBLAST. The alignment program CLUSTALW (http://www.ebi.ac.uk/clustalw/) was used for the nucleotide comparative studies. Phylogenetic trees were created by Geneiuos Tree Builder using the following parameters: genetic distance model, Juke-Cantor; tree build method, Neighbor- Joining; 4 DAPI/CARD-FISH. DAPI staining procedure was performed along with Fluorescence In Situ Hybridization as described in the lab manual. 100 µL of each secondary enrichment was filtered through a 0.2 µm filter. After cutting each filter in 8 pieces, one piece was embedded with DAPI staining for counting the entire number of cells present in the enrichment. The other pieces were treated with one of the following probe: - Alf986 (5’-GGTAAGGTTCTGCGCGTT-3’) - Bet42a (5’-GCCTTCCCACTTCGTTT-3’) - Gam42a (5’-GCCTTCCCACATCGTT-3’) - EubI-III (5’-GCWGCCWCCCGTAGGWGT-3’) The formamide concentration in the hybridization buffer was 35% for the treatment of the sample with each probe. An unlabelled target competitor was used in the hybridization reaction involving the probes Bet42a and Gam42a. The competitors were the unlabelled probes Gam42a and Bet42a, respectively. Pigment analysis. Spectra of 1 mL cultures were measured spectrometrically from 350 to 1100 nm. Chemotaxis activity assay. 200 µL of the enrichment under analysis was inoculated inside a chamber created by sealing the edges of a cover slip onto a slide. Five capillaries each containing a different substrate to be tested as chemotaxis inducer were inserted inside the chamber. After 1 hour of incubation the capillaries were observed through a phase-contrast microscope at 100X of magnification. RESULTS Primary and secondary enrichments of anoxygenic phototrophic bacterial communities. Two primary enrichments grew after two weeks from the initial inoculum. The cultures that showed increased turbidity were those containing
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