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.
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01(/2&341*+,5*)657*8% $$# 9&:43,52&341&:5;*'+,:(*5<9%-= >8# 7*8% B:,,)52&341&:5;*'+,:(*5
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 either acetate or succinate as electron donor and that were illuminated at either 960 nm (the one with acetate) or 880 nm (the one with succinate). 500 µL of each of these two enrichments were used as inoculum of a new series of bottles each containing one different electron donor. They were exposed to the same wavelength of light of the first enrichment used as inoculum. The second enrichments that were able to grow (after 10 days) were both including succinate as electron donor. The corresponding cultures were named LSW_880 and LSW_960.
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Fig. 2 The two bottles containing the first enrichments grown at either 960nm or 880nm.
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Fig. 3 The two bottles containing the second enrichments grown at either 960nm or 880nm.
Phylogenetic analysis of the isolates grown in Agar Shake Tubes. After one week of growth, 10 single colonies were isolated from agar shake tube dilution cultures inoculated with either LWS_880 or LWS_960 enrichment (Fig 4) (6 colonies for LWS_960 and 4 colonies for LWS_880).
A B Fig. 4. Grown agar shake tube dilution cultures inoculated with either LWS_880 (A) or LWS_960 (B)
Colony PCR amplification products of both 16S rDNA gene and pufM gene for each isolate are shown in Fig. 5
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A B Fig. 5. Colony PCR product bands obtained by using as primer set either 8F-1492R (A) or PB557F- PB750R (B)
6 The 16S rDNA gene of six isolates was analysed (4 isolates from LWS_880 named as ST_880A, ST_880B, ST_880C and ST_880D and 2 isolates from LWS_960 named as ST_960D and ST_960E). The corresponding phylogenetic tree showed that all the six isolates clustered together and that they were correlated to uncultured members of the Cytophaga- Flavobacterium-Bacteroides taxonomic group. The 16S rDNA gene similarity with the closest reference strains in database was only 94-95% for all the isolates under analysis, as shown in Fig. 6.
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0%1( !"#$%&'"()%*' +','-').'("&-%/) 3#4-.' !2') !"#$%&$'()*+,#&('-$.* 8,#&('-,%*)-9(':-&;*-"*,* 356789: 567 #%/"(*01!02342 <,'-++(,"*#/',% !"#$%&$'()*+,#&('-$.* 8,#&('-,%*)-9(':-&;*-"*,* 356789; 5=7 #%/"(*01!02342 <,'-++(,"*#/',% !"#$%&$'()*+,#&('-$.* 8,#&('-,%*)-9(':-&;*-"*,* 356<<9= 5=7 #%/"(*01!02342 <,'-++(,"*#/',% !"#$%&$'()*8,#&('/-)(&(:* 8,#&('-,%*#/..$"-B(:*/C* 356<<9> +,#&('-$.*#%/"(* 567 D-%)*,")*#,EB9(*:E/"F(*-"* >?<,4@ The pufM gene of nine isolates was also analysed (3 isolates from LWS_880 named as ST_880A, ST_880B and ST_880C, and 6 isolates from LWS_960 named as ST_960A, ST_960B, ST_960D, ST_960E and ST_960F). The phylogenetic tree resulting from the alignment of the pufM genes of the isolates with the reference pufM genes in database showed that some isolates were phylogenetically correlated with members of Rhodobacter genus while other strains were phylogenetically correlated with Marichromatium genus. The comparison of the pufM sequence of the isolates with those in database reveal the presence of purple sulphur bacteria belonging to the genera Thiorhodococcus, Marichromatium and Allochromatium in LWS_960. Purple non-sulphur bacteria were, conversely, showed among the isolates from LWS_880 including strains belonging to Rhodobacter and Rhodospirillum 7 genera. !"#$%&.( !"#--&,( !"#--&.( Max Isolates name Reference strain Taxonomy Iden !"#--&+( Gammaproteobacteria; ST_960A Thiorhodococcus drewsii 97% Chromatiales;Chromatiaceae !"#$%&,( Gammaproteobacteria; ST_960B Marichromatium fluminis 95% Chromatiales;Chromatiaceae Gammaproteobacteria; !"#$%&'( ST_960C Allochromatium renukae 87% Chromatiales;Chromatiaceae Gammaproteobacteria; !"#$%&)( ST_960D Marichromatium fluminis 96% Chromatiales;Chromatiaceae Gammaproteobacteria; !"#$%&*( ST_960E Marichromatium fluminis 95% Chromatiales;Chromatiaceae Gammaproteobacteria; ST_960F Marichromatium fluminis 97% Chromatiales;Chromatiaceae Alphaproteobacteria; !"#$%&+( ST_880A Rhodospirillum rubrum 87% Rhodospirillales; Rhodospirillaceae Alphaproteobacteria; ST_880B Rhodobacter blasticus 86% Rhodobacterales; Rhodobacteraceae Alphaproteobacteria; ST_880D Rhodobacter sphaeroides 87% Rhodobacterales; Rhodobacteraceae Fig. 7. On the left: the phylogenetic tree based on the CLUSTALW alignment of the pufM gene sequences of the isolates with the pufM genes in database. Isolates names: ST_880A, ST_880B, ST_880D for LWS_880 and ST_960A, ST_960B, ST_960C, ST_960D, ST_960E, ST_960F for LWS_960. On the right: the table reports the closest reference strain in database for each isolate based on the pufM gene sequence similarity. Phylogenetic analysis of the isolates grown on agar plates. After two weeks, single colonies growth was visible on the agar plates where 100 µL of the anoxygenic phototroph cultures (LWS_880 and LWS_960) were spread onto separately. On the basis of the different morphology and color, five colonies were analysed from each enrichment. !"#$%&'( A B 8 Fig. 8. Growth on agar plates of single colonies from the enrichments LWS_880 (A) and LWS_960 (B). As for the isolates grown in the Agar Shake Tubes, both 16S rDNA and pufM genes were amplified from each single culture by colony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he analysis of 16S rDNA gene of seven isolates revealed the presence of purple non-sulphur bacteria in both the enrichments. All the isolates from LWS_960 were closely related with Rhodovolum genus members. The 16S rDNA gene of two of the isolates from LWS_880 show high similarity with the following purple non-sulphur bacteria genera: Rhodovolum and Rhodobium. The isolate named as PL_880C was taxonomically correlated with Marinobacter genus belonging to the Alteromonadales order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ig. 10. On the left: the phylogenetic tree based on the CLUSTALW alignment of the pufM gene sequences of the isolates with the pufM genes in database. Isolates names: PL_880A, PL_880B, PL_880C, PL_880D and PL_880E for LWS_880 and PL_960B, PL_960C, PL_960D, PL_960E for LWS_960. On the right: the table reports the closest reference strain in database for each isolate based on the pufM gene sequence similarity. The analysis of pufM gene amplified from 9 of the isolates (4 isolates from LWS_960 and 5 isolates from LWS_880) confirmed the presence of strains belonging to Rhodovolum genus in both the enrichments. Other isolates in both the enrichments had the pufM gene sequences showing the highest similarity with those of uncultured bacteria in database described to compose autotrophic bacterial communities (Perreault N.N., 2008) DAPI/CARD-FISH. Direct microscopic count of the cells present on the filters of each sample was performed by DAPI staining. As a result the measured cell densities were 5.22x107 cells/mL and 2.80x108 cells/mL in the enrichments LWS_880 and LWS_960, respectively. In order to analyse the composition of the enrichments LWS_880 and LWS_960, three different probes were used targeting the three major bacterial groups (α-, β- and γ- proteobacteria) among which purple sulfur and non-sulfur bacteria are distributed. The eubacterial probe EubI-III stained 97% and 96% of the total number of cells on the filters of LWS_880 and LWS_960, respectively. A negative control was also performed using a nonsense probe that allows the detection of non-specific binding. The density of the cells detected by each probe and in each sample are reported in Fig. 11. 10 !"#$%%&' !"#$()&' !"#$" *+,-.' <=>?@A'BCD'E!' *+,-.' !"#$" <=>?@A'BCD'E!' /!*0()%' ;FG;.H&G' /!*0()%' IF&%.H&%' -.1234' IFI3.H&G' -.1234' JFJJ.H&)' .5-67666' 2F&(.H&G' .5-67666' 3F%;.H&%' 8/9234' IF;3.H&G' 8/9234' (F;%.H&G' :,:;;%' &' :,:;;%' &' Fig. 11. The probes utilized in CARD-FISH assays are indicated on the left side of the pictures representing the correspondent hybridization with each enrichment sample (LWS_880 and LWS_960). On the right of each picture the density of the cells detected by the probe per mL of culture is reported. Subsequently, the relative abundance of each bacterial group targeted by the probes was calculated (Fig 12). It was expressed as percentage of the entire number of cells detected by the probe EubI-III. "#$%&'(!)*! "#$%&&'!()! 20%! 32%! 53%! 42%! !"#$%#&'()'*%+()&,%--34/5#/,%'(%0#$'(,1#* 25%! .)(%#&'()'*%+()&,%--2('#/,%'(%0#$'(,1#* 26%! /%00%#&'()'*%+()&,%---#..#/,%'(%0#$'(,1#* 2%! !"#$$%&"'()*+%,* %&#$ Fig. 12. Relative abundances of each group of Proteobacteria in the enrichment LSW_960 (left) and LWS_880 (right). !"#$ The data described in Fig. 11 suggest that the enrichment LSW_880 is evenly distributed %#$ %!#$ among the three proteobacterial group. On the contrary, more than a half (53%) of the cells in LSW_960 is represented by α-proteobacteria while β-proteobacteria are only 2%. Interestingly, 20% of the whole bacterial fraction has not been targeted by any of the proteobacterial probes suggesting the presence in this enrichment of bacteria belonging to 11 other classes. Pigment analysis. The absorption spectra of whole cells were collected for each enrichment. The absorption peaks at 797 and 908 in the spectrum of the sample LWS_880 represent the typical absorption maxima of bacteriochlorophyll a (bchla) suggesting the major presence of purple bacteria having this photosynthetic pigment. (Fig 13) !"#$%%&' ()*+'!' Fig. 13. Absorption spectrum of whole cell sample of the enrichment LSW_880. The absorption peaks at 807 and 911 in Fig 14 also indicate the presence of a bchl a as main photosynthetic pigment in the sample LWS_960. These peaks correspond to those associated with a new type of light-harvesting complex in Roseospirillum parvum described by Glaeser & Overmann (1999). 12%$2%34&4""5+'3#&*5+!"#$%! !"#$%&'(( )*+,(!( !"#$%$&'(')*$&+#,,'-.///0' Fig. 14. Absorption spectrum of whole cell sample of the enrichment LSW_880. Chemotaxis activity assay. The presence of motile cells was observed in both the samples LWS_880 and LWS_960 by observing 100 µL of each enrichment through the phase-contrast microscope at 100X magnification. Nevertheless, chemotaxis activity was shown only by bacteria in the sample LWS_960 towards some of the substrates that were tested. The bacteria 12 showed chemotactic activity mainly towards glycerol but also towards sodium succinate and sodium acetate. No chemotaxis was shown towards either caso-amino acids or sulfide. !##,##3###:###B# !"#$%&'('&#)*+*,-.# ,"#/0%%12('&#)*+*,-.# 3"#456%&785#)*+39.# :"#;(<=$>128$%1?<#)*+39#@A@.# B"#/05C?&#)*+*!-D#EF#G+,.# Fig. 15 Sealed slide containing the chamber where the sample is inoculated and where the capillaries with the substrates to be tested are inserted. DISCUSSION In this study I enriched for the anoxygenic phototroph bacterial communities LWS_880 and LWS_960 that are able to grow at 880 nm and 960 nm, respectively, using succinate as electron donor in the photosynthetic process. The composition of the two communities was assessed by using both molecular and biochemical methods. The culturable bacteria were identified at the molecular level by both inoculating the mixed cultures into Agar Shake Tubes and by spreading them onto Agar plates. The results obtained from the analysis of 16S rDNA gene of the isolates grown in Agar Shake Tubes showed very poor similarity (95%) with members of the group Bacteroidetes/Cytophaga. The inconsistency of these 16S rDNA gene data was confirmed by the analysis of the pufM gene sequences from the same isolates that identified these bacteria as purple sulfur bacteria in LWS_960 and purple non-sulfur bacteria in LWS_880. The anoxygenic phototrophs that grew in Agar Shake tubes were shown to belong to the following purple sulfur bacteria genera: Thioroducoccus, Allochromatium, Marichromatium in the case of LWS_960. The bacteria composing LWS_880 able to grow in Agar Shake Tubes were shown to have pufM gene sequences highly correlated with those of members of purple non- sulphur bacterial genera Rhodospirillum and Rhodobacter. Interestingly, the results obtained from the molecular analysis of 16S rDNA and pufM genes of the isolates grown on Agar Plates indicated the presence of strains belonging to Rhodovolum genus in both the bacterial consortia and to Rhodobium genus only in LWS_880. Complementing the results obtained 13 with the different isolation techniques, the composition of the culturable bacteria of the anoxygenic phototrophic community LWS_880 includes exclusively purple non-sulphur bacteria belonging to the genera Rhodospirillum, Rhodobacter, Rhodbium and Rhodovolum. Conversely, LWS_960 is composed by both purple sulfur bacteria (Thioroducoccus, Allochromatium, Marichromatium genera) and purple non-sulfur bacteria (Rhodovolum genus). The different composition of the two anoxygenic phototroph consortia was also confirmed by CARD-FISH hybridization assay. The consortium LSW_880 was shown to be evenly distributed among the three classes of Proteobacteria while LSW_960 was composed of more than 50% by α-proteobacteria and the β-proteobacteria were only 2% of the total bacterial fraction. 20% of the bacterial biomass in LSW_960 was not detected by any of the three Proteobacteria probes suggesting the presence of bacteria belonging at least to another bacterial phylum. The analysis of the pigments also showed the presence of different light-harvesting complexes in the two anoxygenic phototroph communities. The absorption spectrum of LWS_880 showed the two typical absorption peaks corresponding to bChl a. Interestingly, the absorption peaks shown in the spectrum of LWS_960 sample correspond to those associated to the bChl a in the purple non-sulfur bacterium Roseospirillum parvum containing a new type of photosynthetic light-harvesting (LH) complex that presents an unusual absorption maximum at 911 nm. The great diversity of the pigment-protein complexes in anoxygenic phototrophic bacteria seems to be the result of the strong competition for the wavelengths in the infrared light in the sediment environment (Glaeser & Overmann, 1999). Finally, chemotaxis assay was performed since purple non-sulfur bacterial group is known to have members that show great motility. Only LW_960 showed chemotaxis activity towards glycerol, succinate and acetate. No clear results were obtained for LW_880. In this study molecular and biochemical techniques were applied to characterize two anoxygenic communities grown at different wavelength of light in the IR radiation. Further studies will be focusing in obtaining pure cultures of the isolates in order to perform both biochemical and biophysical analyses of the single cultures of anoxygenic phototrophic molecularly described in this report. 14 BIBLIOGRAPHY Achenbach L.A., Carey J. and Madigan M.T. (2001). 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