1  

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: marty.cap@gmail.com

Microbial Diversity Course

Marine Biological Laboratory, June-July 2011

  2  

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

  3  

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.

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

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  4  

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;

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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.

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

A B Fig. 5. Colony PCR product bands obtained by using as primer set either 8F-1492R (A) or PB557F-

PB750R (B)

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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.

Fig. 6. On the left: The phylogenetic tree based on the RDP analysis of the 16S rDNA sequences of the isolates obtained from the second enrichments LWS_880 (isolates names: ST_880A, ST_880B, ST_880C, ST_880D) and LWS_960 (isolates names: ST_960D and ST_960E) (Streptomyces sp. is the outgroup). On the right: the table reports the closest reference strain in database for each isolate based on the 16S rDNA gene sequence similarity. .

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

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genera.

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.

 

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Isolates name Reference strainMax Iden Taxonomy

ST_960A Thiorhodococcus drewsii 97% Gammaproteobacteria; Chromatiales;Chromatiaceae

ST_960B Marichromatium fluminis 95% Gammaproteobacteria; Chromatiales;Chromatiaceae

ST_960C Allochromatium renukae 87% Gammaproteobacteria; Chromatiales;Chromatiaceae

ST_960D Marichromatium fluminis 96% Gammaproteobacteria; Chromatiales;Chromatiaceae

ST_960E Marichromatium fluminis 95% Gammaproteobacteria; Chromatiales;Chromatiaceae

ST_960F Marichromatium fluminis 97% Gammaproteobacteria; Chromatiales;Chromatiaceae

ST_880A Rhodospirillum rubrum 87%Alphaproteobacteria; Rhodospirillales; Rhodospirillaceae

ST_880B Rhodobacter blasticus 86%Alphaproteobacteria; Rhodobacterales; Rhodobacteraceae

ST_880D Rhodobacter sphaeroides 87%Alphaproteobacteria; Rhodobacterales; Rhodobacteraceae

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  9  

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 PCR.

Fig. 9. On the left: The phylogenetic tree based on the RDP analysis of the 16S rDNA sequences of the isolates obtained from the enrichments LWS_880 (isolates names: PL_880A, PL_880C, PL_880D, PL_880E) and LWS_960 (isolates names: PL_960A, PL_960C and PL_960D) (Streptomyces sp. is the outgroup). On the right: the table reports the closest reference strain in database for each isolate based on the 16S rDNA gene sequence similarity. The 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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Fig. 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.

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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.

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

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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).

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

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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.

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

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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.

  15  

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