7. ANNEXES
7.1. Unpublished data
7.1.2. Expression of cbbL and cbbM directly in Lake Cadagno (in situ)
The expression of both RuBisCO genes (cbbL and cbbM) of Candidatus “T. syntrophicum”
strain Cad16T were also fallowed by qRT-PCR (see Material and Methods of Chapter 2) in dialysis bags pure cultures incubated in the chemocline of the Lake Cadagno (see Material and Methods of Chapter 2). The aim of this analysis was to assess whether transcription profiles of Cad16T cells grown in laboratory conditions (i.e., in vitro) were similar to those of bacteria incubated in the Lake Cadagno chemocline (i.e., in situ). For mRNA expression analysis, 100 ml of pure cultures of PSB Candidatus T. syntrophicum strain Cad16T were incubated for five weeks (August 20 to September 24, 2009) in eight 30 cm long dialysis bags (same characteristics as above) at a depth of 12 m. Cells were then sampled every 4 h for a 24 h period, starting at 3:00 PM on September 23. In this period of the year, the sun shone over the lake from 7:48 to 18:34 affording a period of light-exposure 74 min shorter than the 12 h photoperiod selected for the in vitro experiments. Interestingly, the transcription profiles of cbbM in Cad16T cells grown in laboratory conditions or incubated in situ differed considerably, with a single peak of cbbL synthesis measured in T4 = 3.39x10-6 pg of mRNA per cell during the night at 23:00.
On the other hand, the expression of cbbM displayed similar levels of expression compared with those recorded in laboratory conditions (between 10-7 and 10-8 pg of mRNA per cell), without any major peak of expression.
Figure A3. Expression of cbbL and cbbM in situ
Expression of the cbbL gene (full square) and cbbM (empty square) incubated in dialysis bags in the chemocline of the Lake Cadagno of pure Cad16 cultures were monitored every 4 hours for 24 h by RT-qPCR. The sampling points were as follows: T0 = 7:00, T1 = 11:00, T2 = 15:00, T3 = 19:00, T4 = 23:00 and T5 = 3:00. The quantity of mRNA was normalized to the cell number to obtain a direct measurement of the relative cell contribution. All the experiments were performed in triplicate.
7.2. DENMARK: FEMS Research Fellowship rapport
7.2.1. Transcriptomic analyses on the purple sulfur bacterium Thiodictyon sp. Cad16
Nicola Storelli1, Niels-Ulrik Frigaard3, Donald A. Bryant4 and Mauro Tonolla1,2
1 Cantonal Institute of Microbiology, Microbial Ecology Group, Microbiology Unit, Plant biology Dept. UNIGE, Via Mirasole 22a, CH-6500 Bellinzona
2 Centro di Biologia Alpina (CBA), Piora, CH-6777 Quinto
3 Copenhagen Biocenter, Department of Biology University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen N (Denmark)
4 Department of Biochemistry an Molecular Biology, The Pennsylvania State University, University Park, PA 16802 USA
ABSTRACT
Purple sulfur bacteria (PSB) play an essential role in the turnover of carbon and sulfur in many aquatic ecosystems. Their physiological diversity as photoautotrophs, photoheterotrophs, and sometimes as chemotrophs has been recognized for decades, but the significance of this diversity in natural environments is not known. The genome of Thiodictyon sp. Cad16 has been sequenced using pyrosequencing. Preliminary analyses indicate that the genome is unexpectedly large, at least 8 Mbp, and contains approximately 8000 genes with encoding functions related to anoxic, oxic, phototrophic, and chemotrophic lifestyles. This study aimed to investigate the physiology of the PSB Thiodictyon sp. Cad16, which is abundant in our model ecosystem (Lake Cadagno, Switzerland) and grows readily in pure culture. We produced transcriptomic sequence information to correlate changes and adaptations in physiology with changes in environmental conditions. To this end, cultures of Thiodictyon sp. Cad16 were grown under different, defined laboratory conditions (anoxic photoautotrophy, anoxic photoheterotrophy, anoxic chemotrophy, oxic chemotrophy, etc.), mRNA was isolated and cDNA prepared. The cDNA was then sequenced using Illumina’s Solexa approach and compared to the whole genome sequence.
INTRODUCTION
Lake Cadagno is a crenogenic, meromictic lake located in the Piora valley in the southern Alps of Switzerland (46°33' N, 8°43'E). It is characterized by a compact chemocline with high concentrations of sulfate; steep gradients of oxygen, sulfide, and light; and a turbidity maximum that correlates to large numbers of bacteria (up to 107 cells ml-1) (Tonolla, Demarta et al. 1999;
Luthy, Fritz et al. 2000; Camacho, Erez et al. 2001). The chemocline of the meromictic Lake Cadagno contains a dense community of phototrophic purple sulfur bacteria (PSB) belonging to the genera Lamprocystis, Thiocystis, Thiodictyon and Chromatium, as well as the green sulfur bacteria (GSB) of the genus Chlorobium (Tonolla, Demarta et al. 1998; Tonolla, Demarta et al.
1999).
Currently, no genome sequence is available for any PSB from freshwater environments.
Therefore, we decided to sequence the genome of the Thiodictyon sp. strain Cad16 isolated from Lake Cadagno and usually growing in pure cultures (Peduzzi and Tonolla, data not published).
This PSB is currently being sequenced at the laboratory of D.A. Bryant, Pennsylvania, USA, using a pyrosequencing technology approach (Margulies, Egholm et al. 2005). Preliminary analyses indicate that the genome is unexpectedly large, at least 8 Mbp, and contains approximately 8000 genes encoding functions related to anoxic, oxic, phototrophic, and chemotrophic lifestyles.
PSB, similar to GSB, play an essential role in the turnover of carbon and sulfur in many aquatic ecosystems (Gemerden 1986; Camacho and Vicente 1998; Camacho, EREZ et al. 2001).
Their physiological diversity as photoautotrophs, photoheterotrophs, and sometimes as chemotrophs has been recognized for decades (Brune 1995), but the significance of this functional diversity in natural environments is not known.
We expect the outcome of this project to be of importance in the understanding of the physiological capabilities and adaptation strategies of PSB in natural environments and should provide the basis for the annotation of the genome sequence.
MATERIALS AND METHODS Bacterial strain and growth condition
Thiodictyon sp. Cad16 was grown in liquid medium (Pfenning's Medium I, by DSMZ;
hereafter also referred to as “PSB medium”) containing 0.5 g KH2PO4 liter-1, 0.34 g NH4Cl liter
-1, 0.5 g MgSO4 · 7H2O liter-1, 0.25 g CaCl2 · 2H2O liter-1, 0.34 g KCl liter-1, 1.5 g NaHCO3 liter
-1, 0.5 ml of trace element solution SL10, and 0.02 mg of vitamin B12 (Eichler and Pfennig 1988).
The medium was reduced with 0.3 g liter-1 Na2S · 9H2O (1.10 mM final concentration) and adjusted to approx. pH 7.2. Media were prepared in a 2-liter bottle with an N2/CO2 (80%/20%) gas phase according to Widdel and Bak (Widdel and Bak 1992). All cultures were incubated at room temperature (20 to 25°C) with a 12 h light/12 h dark photoperiod at light intensities of 4 to 8 µE m-2 s-1, generated with an incandescent 60-W bulb (Eichler and Pfennig 1988). The growth of the cultures was monitored at a wavelength of 650 nm in a UV/VIS Spectrometer Lambda 2S (Perkin-Elmer) with a 1-cm cuvette.
DNA and RNA analysis
Total mRNA extraction. Four tubes containing 2 ml of Cad16 liquid culture (OD650 0.5< x <1.0) were harvested after 10 min centrifugation at 7,000 g. Then, 0.25 ml of TRIZOL was added to the pellets in the tubes. The material was gently shaken for few minutes to resuspend and lyse the cells, and finally, all samples were collected in one 1.5 ml Eppendorf tube. We extracted the RNA and resuspended it in a 1 ml Eppendorf tube containing approximately 1 ml of TRIZOL-cell TRIZOL (Invitrogen™l). In addition, 0.2 ml of chloroform was added and after a rapid resuspension and incubation for 2-3 min at room temperature, the suspension was centrifuged for 15 min at 12,000 g at 4°C. The aqueous phase was then transferred into a 1.5 ml RNase-free tube, purified on ice with isopropanol and ethanol 70%, and centrifuged twice for 10 min at 12,000 g and 4°C. The RNA was then air-dried for 5-10 min and resuspended in 50 µl of RNA-free water. The RNA was stored at -80°C or used for the next step of purification.
DNase treatment. The RNA extracted was purified using the “TURBO DNA-freeTM kit”
(Ambion®) following the manufacturer’s instructions.
RNA concentration. The purified RNA was concentrated using the “RNeasy MinElute Cleanup kit” (Qiagen®) following the manufacturer’s instructions.
During the purification procedures with this kit, we lost a large amount of RNA. Therefore, we decided to skip this step and concentrate the 50 µl recovered after DNase treatment with a SpeedVAC to a volume of 10-15 µl (approximately 20-30 min).
mRNA enrichment. The concentrated RNA was treated with a “MICROBExpressTM kit”
(Ambion®), following the manufacturer’s instructions to remove the 16S and 23S ribosomal RNAs (rRNA and tRNA).
Amplification of mRNA. The mRNA was transformed in aRNA (mRNA with a PAP tail in the 3’ extremity) and amplified using a “MessageAmp II-Bacteria kit” (Ambion®), following the manufacturer’s instructions.
Double-stranded cDNA synthesis from aRNA. The synthesis of cDNA was carried out using the “SuperScriptTM Choice System for cDNA Synthesis kit” (InvitrogenTM), following the manufacturer’s instructions.
Ligation of all cDNA fragments. The fragments of cDNA were ligated with T4 DNA ligase (Biolabs, M02025). This step stabilizes the cDNA for the next sequencing step. Then, 1 µl of T4 DNA ligase was added to a final reaction volume of 20 µl, which contained 4 µl of 10x buffer provided with the T4 DNA ligase, and incubated overnight at 16°C. After the reaction, the sample was incubated for an additional 20 min at 65°C to inactivate the enzyme and then stored at -80°C.
cDNA sequencing
The cDNA will be sent to another laboratory to be sequenced using Illumina’s Solexa approach. At present, we are evaluating the best offers.
Protein collection
The pellets harvested by centrifugation for 20 min at 9,000 g in two 50 ml Falcon tubes from approximately 80 ml of liquid culture with an OD650 between 0.5 and 1.0 were resuspended in 4 ml of KH2PO4 0.1 M and aliquoted in six 1.5 ml Eppendorf tubes. The 6 tubes were centrifuged for 10 min at 17,500 g, and the supernatant was discarded and the pellets stored at –80°C. All centrifugation steps were performed at 4°C.
RESULTS
Choice of the different growing conditions
The techniques used for the growth in vitro of the PSB is completely different from those currently employed for common bacteria such as E. coli or Pseudomonas aeruginosa. PSB is anaerobic, therefore we have to utilize special, hermetically sealed containers with anaerobic conditions (1:5 - CO2:N2); the cultivation in plates is most problematic. Liquid cultures of Thiodictyon sp. Cad16 need between 6 and 7 days to reach a reasonable optic density at standard heterotrophic growth condition (OD650 of 0,5-1,0). Despite the growth problems with our bacteria, we were able to grown it at 7 different conditions at which the cultures reached a reasonable OD650, but for some of them, detailed below, a pre-incubation during approximately 5 days at standard heterotrophic growth conditions were necessary to minimize the incubation time:
heterotrophy
heterotrophy in continuous light
heterotrophy with oxygen (microaerophily) *
heterotrophy with oxygen in the dark (chemoheterotrophy) * heterotrophy in the dark *
autotrophy
autotrophy with oxygen in the dark (chemoautotrophic) *
* For growth in the presence of oxygen under continuous darkness, the culture was incubated first under normal conditions (atmosphere 1:5 CO2:N2 and with light cycles of 12 hours light/dark) and then for 2-3 more days at special conditions.
More accurate explanations of each growth condition are described in the appendix A at the end of the rapport. From these conditions mRNA was isolated and cDNA prepared. This cDNA will be sequenced using Illumina’s Solexa approach.
mRNA isolation and cDNA preparation
The preparation of the ligated cDNA from the liquid cultures was carried out using more specific kits, as previously explained. After each purification and translation step a quantification
of the mRNA or cDNA was performed using a NanoDrop spectrophotometer. The nucleic acid concentration is presented in Table F1.
Table F1. Nucleic acid concentration.
*2.0 µg is the minimum amount of cDNA needed for a sequencing analysis;
** growth conditions detailed in the appendix A;
*** for each purification/translation step some aliquots were stored at -80°C as reserve.
The different yield between the growth conditions was imputable to essentially 2 reasons, the first was that the starting OD650 were not always the same, the second that I carried out the experiment of the aRNA synthesis in 2 times and with 2 different MessageAmp II-Bacteria kits.
The first one yielded less aRNA (5, 8 and 10 in Table 1.) than the new one. The aRNA of samples 1 and 2 were synthesized with both kits (old and new) because they were grown at the 2 most important growth conditions (heterotrophy and autotrophy).
Collection of protein
Parallel to the mRNA manipulation, we stored a pellet derived from the centrifugation of about 80 ml of liquid culture aliquoted in six 1.5 ml Eppendorf tubes at –80°C. This pellet will be used for proteomic analysis to be carried out using 2D-gel electrophoresis (DIGE) and the results will be correlated to genomic and transcriptomic analysis.
DISCUSSION
The host laboratory headed by Dr. Niels-Ulrik Frigaard has extensive experience with bioinformatics and the functional genomics of GSB (Eisen, Nelson et al. 2002) and has recently moved into the field of PSB. This move is the result of a collaboration with Dr. Tonolla.
Bioinformatics analyses of the PBS genome sequence data will allow us to learn more about the physiology of these organisms, which are an important part of the ecosystem of Lake Cadagno (Tonolla, Peduzzi et al. 2005).
My work in Denmark was essentially to grow the PSB Thiodyction sp. Cad16 in all possible conditions and to collect the largest variety of mRNA. This total mRNA was later converted into cDNA, and all of the fragments were ligated together to form a large cDNA molecule. This cDNA is more stable for storage and can be sequenced more easily. We now shall send our cDNA to the best laboratory (quality-price) for sequencing The sequencing results will then be compared with sequences present in Internet databases (NCBI, etc.), thus enabling us to identify a large part of genes presents in the genome. After the genomic and transcriptomic analyses, we shall conduct some proteomic experiments with 2D-gel electrophoresis (DIGE) to better understand the life of this PSB.
REFERENCES
Brune, D., Ed. (1995) Sulfur compounds as photosynthetic electron donors. In: Anoxygenic Photosynthetic Bacteria Kluwer Academic Publishers Dordrecht/The Netherlands
Camacho, A., J. EREZ, et al. (2001) Microbial microstratification, inorganic carbon photoassimilation and dark carbon fixation at the chemocline of the meromictic Lake Cadagno(Switzerland) and its relevance to the food web. Aquatic sciences 63, 91-106.
Camacho, A., J. Erez, et al. (2001) Microbial microstratification, inorganic carbon photoassimilation and dark carbon fixation at the chemocline of the meromictic Lake Cadagno (Switzerland) and its relevance to the food web.Aquatic Sciences-Research Across Boundaries 63, 91-106.
Camacho, A. and E. Vicente (1998) Carbon photoassimilation by sharply stratified phototrophic communities at the chemocline of Lake Arcas(Spain). FEMS Microbiol Ecol 25, 11-22.
Eichler, B. and N. Pfennig (1988) A new purple sulfur bacterium from stratified freshwater lakes, Amoebobacter purpureus sp. nov. Arch Microbiol 149, 395-400.
Eisen, J., K. Nelson, et al. (2002) The complete genome sequence of Chlorobium tepidum TLS, a photosynthetic, anaerobic, green-sulfur bacterium. Proceedings of the National Academy of Sciences 99, 9509-9514.
Gemerden, H. (1986) Production of elemental sulfur by green and purple sulfur bacteria. Arch Microbiol 146, 52-56.
Luthy, L., M. Fritz, et al. (2000) In situ determination of sulfide turnover rates in a meromictic alpine lake. Appl Environm Microbiol 66, 712-717.
Margulies, M., M. Egholm, et al. (2005) Genome sequencing in microfabricated high-density picolitre reactors. Nature 437, 376-380.
Tonolla, M., A. Demarta, et al. (1998) Microscopic and molecular in situ characterization of bacterial populations in the meromictic Lake Cadagno. In: Documenta Istituto italiano di.
Idrobiologia Pallanza.
Tonolla, M., A. Demarta, et al. (1999) In situ analysis of phototrophic sulfur bacteria in the chemocline of meromictic Lake Cadagno (Switzerland). Appl Environm Microbiol 65, 1325-1330.
Tonolla, M., R. Peduzzi, et al. (2005) Long-Term population dynamics of phototrophic sulfur bacteria in the chemocline of Lake Cadagno, Switzerland. Appl Environm Microbiol 71, 3544-3550.
Widdel, F. and F. Bak (1992) Gram-negative mesophilic sulfate-reducing bacteria. The Prokaryotes 4, 3352-3378.
7.2.2. APPENDIX A: Growth conditions
Explanation of the codes used on tubes (growth condition):
12h L/D: heterotrophic growth conditions with 2 mM of acetate added to a basic PSB medium; normal condition of light exposure with a photoperiod of 12 hours of light followed by 12 hours of darkness during 7 days; starting OD650=0.589 (in a 1ml cuvette) at 10:00 am.
12h L/D ØAC: autotrophic growth conditions in basic PSB medium; normal condition of light exposure with a photoperiod of 12 hours of light followed by 12 hours of darkness during 8 days;
starting OD650=0.483 (in a 1ml cuvette) at 10:00 am.
24h DO2: heterotrophic growth conditions with 2 mM of acetate added to a basic PSB media;
first at normal light condition with a photoperiod of 12 hours of light followed by 12 hours of darkness during 6 days to an OD650=0.579 (in a 1ml cuvette), and then changing the atmosphere by adding 5% oxygen and storing the sample in complete darkness during 1 day (chemoheterotrophic growth conditions); starting OD650=0.704 (in a 1ml cuvette) at 2:00 p.m..
24h DO2 ØAC: : autotrophic growth conditions in basic PSB medium; first at normal condition of light with a photoperiod of 12 hours of light followed by 12 hours of darkness during 6 days to an OD650=0.364 (in a 1ml cuvette), and then changing the atmosphere by adding 5% oxygen and storing the sample in complete darkness during 1 day (chemoautotrophic growth conditions); starting OD650=0.473 (in a 1ml cuvette) at 2:00 p.m..
24h L: heterotrophic growth condition with 2 mM of acetate added to basic PSB medium;
special conditions of light with a photoperiod of 24h continuous light during 8 days; starting OD650=0.317 (in a 1ml cuvette) at 3:30 p.m..
12h L/D O2: heterotrophic growth condition with 2 mM of acetate added to basic PSB medium; normal conditions of light exposure with a photoperiod of 12 hours of light followed by 12 hours of darkness during 7 days (OD650=0.573), and then changing the atmosphere by adding 5% oxygen and incubating for one more day; starting OD650=1.088 (in a 1ml cuvette) at 3:30 p.m..
8h D: : heterotrophic growth conditions with 2 mM of acetate added to basic PSB medium;
normal conditions of light exposure with a photoperiod of 12 hours of light followed by 12 hours of darkness during 7 days; starting OD650=0.584 (in a 1ml cuvette); sampling after 8 hours of darkness (03:00 am).
8h D ØAC: autotrophic growth conditions in basic PSB medium; normal conditions of light exposure with a photoperiod of 12 hours of light followed by 12 hours of darkness during 7 days;
starting OD650=0.467 (in a 1ml cuvette); sampling after 8 hours of darkness (03:00 am).
8h L: heterotrophic growth conditions with 2 mM of acetate added to basic PSB medium;
normal conditions of light exposure with a photoperiod of 12 hours of light followed by 12 hours of darkness during 7 days; starting OD650=0.795 (in a 1ml cuvette); sampling after 8 hours of light phase (15h003:00 p.m.; 12 hours after the 8h D, same culture).
8h L ØAC: autotrophic growth conditions in basic PSB medium; normal conditions of light exposure with a photoperiod of 12 hours of light followed by 12 hours of darkness during 7 days;
starting OD650=0.437 (in a 1ml cuvette); sampling after 8 hours of light phase (3:00 p.m.; 12 hours after the 8h D, same culture).
24h D: heterotrophic growth conditions with 2 mM of acetate added to basic PSB medium;
normal conditions of light exposure with a photoperiod of 12 hours of light followed by 12 hours of darkness during 7 days (OD650=1.040), and then storing the sample in complete darkness during 3 day; starting OD650=1.105 (in a 1ml cuvette) at 4:30 p.m..
Figure F1. Schematized growth conditions.