Thesis
Reference
Role of phototrophic sulfur bacteria from the chemocline in the primary production of Lake Cadagno
STORELLI, Nicola
Abstract
Phototrophic sulfur bacteria are important for primary production in many stratified lakes. In Lake Cadagno, these bacteria greatly contribute to the total primary production with high values of CO2 fixation both in the presence and absence of light. The small-celled PSB Candidatus “Thiodictyon syntrophicum” Cad16T was the strongest CO2 assimilator and used as model organism. The draft genome sequence of strain Cad16T revealed the presence of two RuBisCO genes (cbbL and cbbM), which were deferentially expressed. 2D-DIGE analysis showed the presence of 23 protein spots up-regulated in the light, and 17 in the dark. Among the 23 protein spots that were up-regulated in the light, three are involved in the storage mechanism that produces granules of poly(3-hydroxybutyrate) from an excess of reducing power and carbon compounds. Among the 17 protein spots up-regulated in the dark, three were found to be part of the autotrophic dicarboxylate-hydroxybutyrate (DC/HB) cycle.
STORELLI, Nicola. Role of phototrophic sulfur bacteria from the chemocline in the primary production of Lake Cadagno. Thèse de doctorat : Univ. Genève, 2014, no. Sc.
4646
URN : urn:nbn:ch:unige-349153
DOI : 10.13097/archive-ouverte/unige:34915
Available at:
http://archive-ouverte.unige.ch/unige:34915
Disclaimer: layout of this document may differ from the published version.
1 / 1
Unité de microbiologie Dr. Mauro Tonolla
Role of Phototrophic Sulfur Bacteria from the Chemocline in the Primary Production of Lake Cadagno
THÈSE
présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie
par Nicola Storelli
de Losone (TI)
Thèse n° 4646
GENÈVE
Atelier de reprographie ReproMail 2014
PUBLISHED PAPERS
†Peduzzi S., †Storelli N., Welsh A., Peduzzi R., Hahn D., Perret X., Tonolla M. (2012) Candidatus "Thiodictyon syntrophicum", sp. nov., a new purple sulfur bacterium isolated from the chemocline of Lake Cadagno forming aggregates and specific associations with Desulfocapsa sp. Systematic and Applied Microbioogyl 35, 139-144.
(DOI: 10.1016/j.syapm.2012.01.001). †: equally contributed.
Storelli N., Peduzzi S., Saad M., Frigaard N-U., Perret X., Tonolla M. (2013) CO2 assimilation in the chemocline of Lake Cadagno is dominated by a few types of phototrophic purple sulfur bacteria. FEMS Microbiology Ecology 84(2), 421-432.
(DOI: 10.1111/1574-6941.12074).
Storelli N., Peduzzi S., Saad M., Frigaard N-U., Perret X., Tonolla M. (2014).
Autotrophic carbon dioxide assimilation mechanism in the dark disclosed by proteome analysis of the purple sulfur bacterium Candidatus “Thiodictyon syntrophicum” strain Cad16T. EuPA Open Proteomics 2, 17-30
(DOI: http://dx.doi.org/10.1016/j.euprot.2013.11.010).
REMERCIEMENTS
En premier lieu je tiens à remercier mon directeur de thèse CC Dr. Mauro Tonolla et co- directeur MER Dr. Xavier Perret de m’avoir permis d’entreprendre ce projet à l’Université de Genève, et au Laboratoire d’écologie microbienne de Bellinzona. Merci également au Prof.
Michel Goldschmidt-Clermont professeur responsable pour cette thèse. Un remerciement spécial va au Professeur associé. Niels-Ulrik Frigaard pour avoir accepté de faire partie du jury de thèse et au Dr. Maged Saad pour son soutien scientifique tout au long de ce travail. Leur aide et leur expérience ont joué un rôle fondamental dans la réussite de cette thèse.
J’aimerais remercier tous les collaboratrices et les collaborateurs de l'ICM, en particulier le groupe du Laboratoire de Biosécurité et le groupe du Laboratoire d'Ecologie Microbienne pour leur aide. Un grand merci encore au Dr. Maged Saad pour avoir partagé ses connaissances sur la protéomique, au Dr. Sandro Peduzzi et au Dr. Paola Gandolfi-Decristophoris pour l’aide au niveau de la cultivation des microorganismes anaerobiques, et au Dr. Valeria Guidi, à Francesco Danza, à Anna Mariotti-Nessurini et au Dr. Damiana Ravasi pour toutes les discussions intéressantes que nous avons eues au cours de ces années.
Ma reconnaissance s’adresse également aux personnes que j’ai connues et m’ont gentiment aidé lors de mes sejours en Danmark à l’Université de Copenhague (Biocenter Department of Biology Section for marine biology): Carina Holkenbrink, Jørgen Deiker Petersen, Chizuko Sakamoto, Bjørn Sindballe Broberg, Tonny D. Hansen; et dans les laboratoires de l’Unité de Microbiologie à Geneve: Dr. Cristina Andrés-Barrao, Dr. Antoine Huyghe, Natalia Giot, Coralie Fumeaux, Dr. Nadia Bakkou, Anissa Ravez, Vanesa Miguelez De La Torre.
Je remercie la Fondation du Centre de Biologie Alpine de Piora et son président Prof. Dr. R.
Peduzzi pour le soutien et la mise à disposition des infrastructures nécessaires aux travaux sur le lac de Cadagno ainsi que la Fédération des sociétés européennes de microbiologie (FEMS) et la Societé de Microbiologie Suisse (SMS/SGM) en particulier son ancien president Professeur Dr.
Dieter Haas pour le soutien financier pendant mon sejour en Danmark.
Je tiens enfin à exprimer mon immense gratitude à Alice Benzoni et ma famille pour m’avoir continuellement soutenu durant cette période.
TABLE OF CONTENTS
REMERCIEMENTS ... iv
SUMMARY ... x
RÉSUMÉ... xii
ABBREVIATIONS ... xvi
LIST OF TABLES AND FIGURES ... xx
1. INTRODUCTION ... 2
1.1. Meromixis ... 2
1.1.1. The crenogenic meromictic Lake Cadagno ... 4
1.1.2. Biota of the Lake Cadagno... 6
1.2. Phototrophic sulfur bacteria ... 8
1.2.1. The sulfur cycle ... 8
1.2.2. Ecological distribution of phototrophic sulfur bacteria ... 10
1.2.3. Photosynthetic inorganic carbon fixation in PSB and GSB ... 11
1.2.4. Phototrophic sulfur bacteria in the Lake Cadagno ... 15
1.3. Proteomics ... 19
1.3.1. Techniques for separating proteins ... 19
1.3.2. Protein identification using mass spectrometry ... 20
1.4. Aims of the PhD thesis ... 21
2. RESEARCH PAPER 1 ... 24
CO2 assimilation in the chemocline of Lake Cadagno is dominated by a few types of phototrophic purple sulfur bacteria ... 24
2.1. Supporting information ... 40
SM1. Recipe of trace elements SL10 and SL12. ... 40
SM2. Dissolved inorganic carbon (DIC) and pH from dialysis bags. ... 41
SM3. Genome analysis. ... 42
References supporting information ... 43
3. RESEARCH PAPER 2 ... 48
Candidatus “Thiodictyon syntrophicum”, sp. nov., a new purple sulfur bacterium isolated from the chemocline of Lake Cadagno forming aggregates and specific
associations with Desulfocapsa sp. ... 48
3.1. Supplementary material S1 (Material and methods) ... 58
Enrichment and cultivation of strain Cad16T ... 58
Phyologenetic analysis with 16S rRNA ... 58
MALDI-TOF MS analysis ... 58
Pigment analysis ... 59
3.2. Supplementary material S2 ... 60
Description of Candidatus “Thiodictyon synthrophicum” sp. nov. strain Cad16T. ... 60
References supporting information ... 61
4. RESEARCH PAPER 3 ... 64
Proteomic analysis of the purple sulfur bacterium Candidatus “Thiodictyon syntrophicum” strain Cad16T isolated from Lake Cadagno ... 64
5. DISCUSSION ... 82
5.1. Primary production by anoxygenic bacteria ... 83
5.1.1. Contribution of phototrophic sulfur bacteria to the primary production ... 83
5.1.2. CO2 fixation in meromictic lakes in absence of light ... 84
5.1.3. Autotrophic inorganic carbon fixation pathways ... 84
5.2. Phototrophic sulfur bacteria in the meromictic Lake Cadagno ... 86
5.2.1. Composition of the phototrophic sulfur bacterial population in the chemocline 86 5.2.2. Capacity of PSB to fix carbon ... 87
5.2.3. Candidatus “Thiodictyon syntrophicum” strain Cad16T ... 88
5.3. Proteomic analysis of Candidatus “T. syntrophicum” strain Cad16T ... 90
5.3.1. Metabolism of Cad16T when grown in the presence of light ... 90
5.3.2. Metabolism of Cad16T in absence of light ... 92
5.4. Conclusions and perspectives ... 96
6. REFERENCES ... 100
7. ANNEXES ... 118
7.1. Unpublished data ... 118
7.1.1. Detection of putative RuBisCO proteins by western blot ... 118
7.1.2. Expression of cbbL and cbbM directly in Lake Cadagno (in situ) ... 123
7.2. DENMARK: FEMS Research Fellowships rapport ... 126
7.2.1. Transcriptomic analyses on the purple sulfur bacterium Thiodictyon sp. Cad16 ... 126 7.2.2. APPENDIX A: Growth conditions ... 135
SUMMARY
Lake Cadagno is a meromictic lake located at 1921 m.a.s.l. in the southern Swiss Alps (46°33’N, 8°43’E) in the catchment area of a dolomite vein rich in gypsum (Piora-Mulde). The chemistry of the lake is directly influenced by the particular geology of this valley. The water that percolates through these erodible sedimentary rocks becomes enriched in minerals and enters the lake through underwater springs. The inflow of this denser water produces a stable stratification of the lake and establishes meromixis. The transition zone between the oxygenic mixolimnion and the anoxygenic monimolimnion that is rich in salts is known as the chemocline and is characterized by steep gradients of oxygen, sulfide, and light. Generally, the chemocline that is positioned at a depth of approximately 12 m coincides with the presence of a dense community of anaerobic phototrophic sulfur bacteria of up to 107 cells ml-1 in summer. This community includes purple sulfur bacteria (PSB; family Chromatiaceae) of the genera Chromatium, Lamprocystis, Thiocystis and Thiodictyon and green sulfur bacteria (GSB; family Chlorobiaceae) of the genus Chlorobium. These phototrophic microorganisms fix inorganic carbon (CO2) via anaerobic photosynthesis using reduction equivalents from reduced sulfur compounds and sunlight as an energy source.
Phototrophic sulfur bacteria are important for primary production in many stratified lakes. In Lake Cadagno, these bacteria greatly contribute to the total primary production with high values of CO2 fixation both in the presence and absence of light. Rates of CO2 assimilation of the most abundant phototrophic sulfur bacteria of the chemocline were measured by 14CO2 quantitative assimilation in dialysis bags (in situ) (Chapter 2). These results indicated that the most efficient fixer was the small-celled PSB Candidatus “Thiodictyon syntrophicum” with values of fixed- CO2 as high as 0.6 (± 0.1) pg of 14C cell-1 in presence of light and ca. 0.4 (± 0.1) pg of 14C cell-1 in the dark. In contrast, Chlorobium clathratiforme that accounted for up to 95% of all phototrophic cells, showed extremely low levels of CO2 fixation.
Candidatus “T. syntrophicum” strain Cad16T was proposed as the type strain of a new species within the genus Thiodictyon (Chapter 3), and used as a model organism for further analyses.
When cultivated in vitro in light/dark cycles of 12 hours, pure cultures of strain Cad16T exhibited the highest 14CO2 fixation during the first four hours of light. Compared to the in situ results, the
14CO2 fixation in the light was similar, while 14CO2 fixation in the dark was found to be lowed to ca. 0.1 (± 0.1) instead to ca. 0.4 (± 0.1) of pg of 14C cell-1. Normally, PSB fix CO2 using the Calvin-Benson-Bassham cycle (CBB cycle), in which the key enzyme is ribulose-1,5- bisphosphate carboxylase oxygenase (RuBisCO). The draft genome sequence of strain Cad16T
revealed the presence of cbbL and cbbM genes, which encode form I and form II of RuBisCO, respectively. Transcription analyses confirmed that, while cbbM remained poorly expressed throughout light and dark cycles, cbbL expression was modulated by light and affected by the available carbon sources (e.g., acetate). Interestingly, cbbL expression did not correlate with the highest levels of CO2 assimilation.
Two-dimensional (2D) difference gel electrophoresis (DIGE) was used to monitor the changes in the proteome of strain Cad16T grown in anoxic autotrophic conditions in presence of light or in the dark (Chapter 4). Using Melanie 7.0 software, approximately 1,000 protein spots were identified amongst which 40 where found to be up- or down-regulated when comparing the two culture conditions. Twenty-three were up-regulated in the presence of light, and 17 were up- regulated in the dark. Among the 23 protein spots that were up-regulated in the light, three are involved in the storage mechanism that produces granules of poly(3-hydroxybutyrate) from an excess of reducing power and carbon compounds. Generally, bacteria use PHB as a reserve for carbon and energy as well as a sink for reducing equivalents. Among the 17 protein spots up- regulated in the dark, three were found to be part of the autotrophic dicarboxylate- hydroxybutyrate (DC/HB) cycle, which is known to be an autotrophic pathway for CO2 assimilation in Archaea. Given all of the above, it is tempting to speculate that the observed CO2 fixation in dark requires compounds synthesized during the day (such as PHB) as energy source and reducing power.
RÉSUMÉ
Le Lac Cadagno est un lac méromictique situé dans la vallée de Piora à 1921 mètres d'altitude, dans la partie sud des Alpes suisses (46° 33'N , 8° 43'E ). La vallée de Piora est très particulière car elle est traversée par une veine de dolomite riche en gypse (Piora - Mulde) qui affecte la chimie du Lac Cadagno. En effet, l'eau qui filtre à travers les roches de dolomite et entre dans le lac par des sources sous-lacustres s’enrichie en minéraux et devient très dense. L'afflux de cette eau plus dense produit une stratification stable en établissant la méromicticité du lac. Entre la couche d’eau plus dense qui se trouve au fond (monimolimnion) et la couche d’eau supérieure (mixolinmion), on trouve une partie où se vérifient des changements très prononcés de gradient d’oxygène, de soufre et de lumière (chemocline). Typiquement, la chemocline se situe à une profondeur d'environ 12 m et à son intérieur se développe une riche communauté de bactéries sulfureuses phototrophes (jusqu'à 107 cellules ml-1 en été). Cette communauté est composée par deux grandes familles: les bactéries pourpres sulfureuses (PSB; famille Chromatiaceae) des genres Chromatium, Lamprocystis, Thiocystis et Thiodictyon et les bactéries vertes sulfureuses (GSB; famille Chlorobiaceae) du genre Chlorobium. Ces micro-organismes phototrophes utilisent des composés réduits du soufre (par exemple: H2S) comme donneurs d'électrons pour la fixation du carbone inorganique (CO2) grâce à l’énergie de la lumière.
Les bactéries sulfureuses phototrophes sont importantes pour la production primaire dans de nombreux lacs stratifiés (méromictiques). Dans le Lac Cadagno, ces bactéries contribuent largement à la production primaire totale avec des valeurs élevées d’assimilation du CO2 soit en présence soit en absence de lumière. Les taux d'assimilation du CO2 des 4 plus grandes populations de bactéries sulfureuses phototrophes de la chemocline ont été mesurés par l’assimilation quantitative de l’isotope radioactif 14CO2 du carbone (Chapitre 2). Ce résultat montre qu’une population est clairement plus active dans l’assimilation du CO2 avec ou sans lumière par rapport aux autres. En effet, la population du PSB Candidatus "Thiodictyon syntrophicum", avec une assimilation d’environ 0,61 (±0,11) pg de 14C cellule-1 en présence de lumière et environ 0,41 (± 0,09) 14C cellule-1 dans l’obscurité, semble être très importante dans la production primaire de la chemocline. Cela est davantage remarquable si l’on considère que la population du GSB Chlorobium clathratiforme, qui domine la chemocline avec environ 95% des cellules phototrophes totales, affiche une assimilation du CO2 extrêmement faible.
La population plus active dans l’assimilation du CO2, le Candidatus "Thiodictyon syntrophicum", a été isolée et caractérisée comme une nouvelle espèce du genre Thiodictyon (Chapitre 3), souche-type Cad16T, et utilisée comme organisme modèle pour des analyses
ultérieures en laboratoire. Des cultures pures de la souche Cad16 ont été faites pousser avec des cycles de 12 heures de lumière suivis par 12 heures d’obscurité en laboratoire (in vitro) afin d’analyser leurs capacités d’assimilation du 14CO2 pendant des tranches de 4 heures. L’activité d’assimilation résulte majeure pendant les 4 premières heures d’exposition à la lumière.
Normalement, les PSB assimilent le CO2 par le cycle de Calvin-Benson-Bassham (cycle de CBB), dans lequel l'enzyme clé est le ribulose-1,5-bisphosphate carboxylase oxygénase (RuBisCO). L’analyse de la séquence brute du génome de la souche Cad16T révèle la présence de deux gènes, cbbL et cbbM, qui codent respectivement pour la forme I et la forme II de l’enzyme RuBisCO. L’analyse de l’expression du mRNA de ces deux gènes pendant une journée (chaque 4 heures) a montré une expression constante de la forme II cbbM, tandis que l’expression de la forme I cbbL semble être influencée par la lumière et les éventuelles souches de carbone (par exemple: acétate) présentes dans le milieu. Dans un milieu autotrophe, chaque 12 heures se vérifient deux pics d’expression du gène cbbL. Ce qui est remarquable est le fait que ces pics ne correspondent pas au moment d’assimilation majeure du 14CO2 montré dans l’expriment précédant (Chapitre 2).
La technique du électrophorèse bidimensionnelle différentielle sur gel utilisant des colorant fluorescents, appelée 2D-DIGE, a été utilisée pour surveiller les changements dans le protéome de Candidatus "T. syntrophicum" souche Cad16T dans un milieu autotrophe en présence ou en absence de lumière (Chapitre 4). Pour chaque expérience de 2D-DIGE, environ 1000 spots de protéines ont été identifiés à l'aide du logiciel spécifique (Melanie 7.0). Parmi ces spots, uniquement 40 satisfont les deux règles (l’ANOVA et l’intensité d’expression minimale) pour indiquer une différence d’expression effective entre les deux situations (lumière vs obscurité). En présence de lumière, 23 protéines étaient plus nombreuses par rapport à l’obscurité, tandis que 17 protéines étaient plus nombreuses dans l’obscurité par rapport à la lumière. Parmi les 23 protéines plus nombreuses en présence de lumière, 3 semblent être impliquées dans un processus qui produit des granules de poly(3-hydroxybutyrate) (PHB), en utilisant des excès de facteurs réduits (NAD[P]H) et des composés de carbone (Acetyl-CoA). Ces composés de réserves constituent une ressource très importante de pouvoir de réduction et d’énergie, étant donné que le carbone stocké peut ensuite être oxydé par le cycle des acides tricarboxyliques (TCA) produisant ATP. Dans l’obscurité, parmi les 17 protéines plus nombreuses, 3 peuvent nous aider à mieux comprendre l’assimilation de CO2 en absence de lumière. Ces 3 protéines font partie du cycle du dicarboxylate-hydroxybutyrate (DC-HB), qui est un cycle autotrophe observé jusqu’à maintenant uniquement chez les Archaea.
Le mécanisme d’assimilation du CO2 en absence de lumière semble être lié à la production de composés de réserve faite en présence de lumière, tels que les PHB ou le glycogène qui stockent le potentiel de réduction et l’énergie nécessaires pour ce mécanisme. Selon nos résultats, les deux cycles, CBB et DC – HB, sont probablement impliqués dans l’assimilation du CO2 dans l’obscurité.
Le mécanisme d’assimilation du CO2 en absence de lumière semble être lié à la production de composés de réserve faite en présence de lumière, tels que les PHB qui stockent le potentiel de réduction et l’énergie nécessaires pour ce mécanisme.
ABBREVIATIONS
2-DE Two-dimensional gel electrophoresis
2D-DIGE Two dimensional fluorescence difference gel electrophoresis 2D-PAGE Two dimensional polyacrylamide gel electrophoresis
ATP Adenosine-5'-triphosphate BChl Bacteriochlorophyll
bp Base pairs
°C Celsius degree
Cad16T Candidatus "Thiodictyon syntrophicum" nov. sp. strain Cad16Type strain
CBB Calvin-Benson-Bassham
14C Radiocarbon, radioactive isotope of carbon CHCA Alpha-cyano matrix solution
DAPI 4′,6-diamidino-2-phenylindole DCF dark CO2 fixation
DC/HB cycle Dicarboxylate/4-hydroxybutyrate cycle
DIC Dissolved inorganic carbon
DNA Deoxyribonucleic acid
DSM Deutsche Sammlung von Mikroorganismen EDTA Ethylenediaminetetraacetic acid
EMBL European Molecular Biology Laboratory FISH Fluoresent in situ hybridisation
GSB Green sulfur bacteria
ha Hectare (10’000 m2)
HP/HB cycle 3-hydroxypropionate/4-hydroxybutyrate cycle HPLC High-performance liquid chromatography
MALDI TOF MS Matrix assisted laser desorption ionization - time of flight mass spectrometry
m a.s.l. Meters above sea level
NAD(P)+ Nicotinamide adenine dinucleotide (phosphate-oxidase) oxided NAD(P)H Nicotinamide adenine dinucleotide (phosphate-oxidase) reduced nanoSIMS Nano-scale secondary-ion mass spectrometry
NJ Neighbor-joining
NPP Net primary production
OD Optical density
ORP Oxidation reduction potential
pBLAST Protein Basic Local Alignment Search Tool PCR Polymerase chain reaction
PFF Peptide fragmentation fingerprinting
pH Power of hydrogen
PHA Polyhydroxyalkanoates
PHB Polyhydroxybutyrate
PMF Peptide mass fingerprinting PNSB Purple nosulfur bacteria
PSB Purple sulfur bacteria
qRT-PCR Reverse transcription-quantitative polymerase chain reaction rRNA Ribosomal ribonucleic acid
rTCA Reverse tricarboxylic acid
RuBisCO Ribulose-1,5-bisphosphate carboxylase oxygenase SARAMIS Spectral archive and microbial identification system
SDS Sodium dodecyl sulfate
SRB Sulfate-reducing bacteria
UV Ultraviolet
LIST OF TABLES AND FIGURES
1. Introduction
p. 2 Table 1. Classification of water stratification regimen.
p. 3 Figure 1. Typical water stratification in a meromictic lakes.
p. 5 Figure 2. The crenogenic meromictic Lake Cadagno.
p. 8 Figure 3. The sulfur cycle.
p. 14 Figure 4. Metabolic pathways for the assimilation of the CO2 in phototrophic sulfur bacteria.
p. 17 Figure 5. Phototrophic sulfur bacteria isolated from the chemocline of the Lake Cadagno.
2. Research paper 1
p. 27 Table 1. Major characteristic of strains used in this study.
p. 28 Table 2. Cy3-labeled oligonucleotide probe used in this study for FISH counting.
p. 30 Figure 1. Vertical profiles of oxygen, sulfide, turbidity, light, ATP, temperature, conductivity, sulfate and oxidation reduction potential (ORP) on 12 september 2007.
p. 31 Figure 2. Mesurements of 14CO2 assimilation by representative GSB and PSB in the chemocline of lake Cadagno.
p. 32 Table 3. Estimated contribution to CO2 fixation by selected groups of organism in lake Cadagno.
p. 32 Figure 3. Variation of 14CO2 assimilation in strain Cad16T under laboratory growth conditions.
p. 33 Figure 4. Levels of strain Cad16T cbbL and cbbM transcripts measured by qRT-PCR.
p. 44 Table SM1. Physical parameters of the Lake Cadagno (12th September 2007).
p. 45 Figure SM1. Specific FISH counts of GSB (white) and PSB (black) compared to the total prokaryotic cells counted by DAPI (grey) at different depths of Lake Cadagno during the day of the 14CO2 assimilation analysis from the cultures pre-incubated in dialysis bags (in situ, September 12, 2007)
3. Research paper 2
p. 51 Figure 1. Phase contrast micrograph of strain Cad16T.
p. 52 Table 1. Differentiating characteristics of species of the genus Thiodictyon and related genera.
p. 53 Figure 2. Maximum likelihood tree topology from 16S rRNA gene sequences for isolate Cad16T and other closely related species of the family Chromatiaceae created using PAUP*4.0b10 and a GTR model of sequence evolution.
p. 54 Figure 3. MALDI-TOF MS dendrogram of 12 strains of phototrophic sulfur bacteria, resulting from single-link clustering analysis.
4. Research paper 3
p. 68 Table 1. CyDye Labeling Scheme and Gel Setup for 2D-DIGE Analysis
p. 69 Figure 1. The protein expression patterns of Candidatus “T. syntrophicum” strain Cad16T separated in a 24 cm, pH 3-10 nonlinear strip and a 12% polyacrylamide gel.
p. 70-71-72 Table 2. List of proteins identified by MALDI-TOF MS/MS
p. 76 Figure 2. Scheme summarizing the majors metabolic pathways suggested from 2D-DIGE analysis.
5. Discussion
p. 88 Figure 6. Incubation of the four major populations of phototrophic sulfur bacteria of Lake Cadagno at 12 m depth using dialysis bags.
p. 93 Figure 7. The dicarboxylate/4-hydroxybutyrate cycle described in Desulfurococcales and Thermoproteales; (B) the 3-hydroxypropionate/4-hydroxybutyrate cycle described in Sulfolobales.
p. 95 Figure 8. (A) The reductive citric acid (rTCA) cycle (B) Cyclic metabolism of PHB biosynthesis and degradation in bacteria
7. Annexes
7.1. Unpublished data
p. 119 Figure A1. Detection of large subunit of the RuBisCO by Western Blot.
p. 120 Figure A2. Western blot against the large subunit from the RuBisCO in a 2D-GEL of proteins extracted during the light exposure.
p. 124 Figure A3. Expression of cbbL and cbbM in situ.
7.2. DANMARK: FEMS Research Fellowships rapport p. 131 Table F1. Nucleic acid concentration. p. 136 Figure F1. Schematized growth conditions.
1. INTRODUCTION
1.1. Meromixis
Vertical stratification in lakes occurs because of differences in densities of water layers, which are mainly influenced by temperature and dissolved substances. In turn, stratification affects pH, dissolved oxygen, nutrient concentrations, light transmission, and the composition of planktonic and benthic organisms (Tonolli 1969; Wetzel 1983; Imboden and Wüest 1995; Pourriot and Meybeck 1995; Wetzel 2001). Lakes normally present two types of stratification: temporal or permanent. Temporary stratification results in holomictic, oligomictic, and polymictic basins that differ in the frequency and intensity of water mixing periods (Table 1). In contrast, meromictic lakes are characterized by a permanent stratification due to incomplete water circulation, resulting in basins in which the lower portion of the water mass never mixes with the rest of the lake. The current classification of such water bodies generally distinguishes three types of meromixis: biogenic, ectogenic and crenogenic (Hutchinson 1937). The biogenic meromixis is due to an intense biological activity that results in an accumulation of dissolved salts and organic material in the lower part of the lake establishment of a stratification regime. Different in the ectogenic meromixis lakes, where the meromictic condition is initiated by some external superficial events as for example the intrusion of water more reach in salts and for this reason more dense, however unless this external event re-occurs the lake change in holomictic after some period of time. In crenogenic meromictic lakes, the stratification is caused by continuous supply of denser saline water by sublacustrin springs. In all cases, the morphometry of lake basin and its topographic position can enhance the stability of the stratification.
Table 1. Classification of water stratification regimen.
Holomictic: complete vertical mixing of the water body, at least once a year;
Oligomictic: irregular, infrequent complete vertical mixing of the water body;
Polymictic: complete vertical mixing more than twice in a year;
Meromictic: permanently stratified water body, incomplete circulation;
The water column of meromictic lakes is generally characterized by three distinct layers (see Figure 1): an upper layer (mixolimnion) that is usually oxic and characterized by a complete circulation of the water during the year; a bottom layer (monimolimnion) that is usually anoxic and characterized by a stagnant water body; and a narrow layer called chemocline that separates the mixo- and monimolimnion and is characterized by steep physical-chemical gradients. Partial mixing events (“meros”) occur in the mixolimnion, which is exposed to the atmosphere, and for this reason, subject to thermal stratifications during summer and mixing events in the cold season.
Figure 1. Typical water stratification in a meromictic lake with the upper oxygenic mixolimnion, the intermediate chemocline and the bottom anaerobic monimolimnion. Each layer harbours specific microoorganisms performing characteristic metabolic functions such as oxygenic photosynthesis and aerobic respiration in the oxic mixolimnion, anoxigenic photosynthesis in the chemocline and anoxigenic respiration in both the chemocline and the monimolimnion.
Because of their permanent stratification, meromictic lakes are interesting objects for microbial ecology, with anoxic sediments representing biological archives of the lake’s history as a result of a stable deposition and the lack of sediment perturbation (Brown and McIntosh 1987; Birch et al. 1996; Coolen and Overmann 1998; Romero et al. 2006; Ravasi et al. 2012;
Wirth et al. 2012). Meromictic lakes also represent ideal models for the study of biogeochemical processes mediated by microorganisms, such as sulfur and nitrogen cycle, due to their stable anaerobic environment find in the monimolimnion (Putschew et al. 1995; Hanselmann and Hutter 1998). In addition, in the chemocline, the physico-chemical gradients and the presence of light support the development of intense blooms of phototrophic sulfur bacteria (Sorokin 1970;
Parkin and Brock 1981; Van Gemerden and Mas 1995; Overmann 1997; Tonolla et al. 1998a;
Hadas et al. 2001; Rodrigo et al. 2001; Koizumi et al. 2004).
1.1.1. The crenogenic meromictic Lake Cadagno
Lake Cadagno is a crenogenic meromictic lake located at 1921 m a. s. l. in the Piora valley in the southern Swiss Alps (46°33’ N, 8°43’ E). The lake is located in a geographic area where ice and snow persist for up to 6 months during the winter season. The lake is approximately 850 m long and 430 m wide, with an average surface area of 26 ha and a maximum depth of 21 m (Del Don et al. 2001). Most probably, Lake Cadagno results from glacial erosion during the last glacial period with an estimated age of 8,000 to 11,000 years (Krige 1918; Del Don et al. 2001;
Wirth et al. 2012). The Piora valley is characterized by the presence of a longitudinal vein of dolomite rock rich in gypsum that influences the chemistry of the Lake Cadagno through sublacustrine springs in its southern part (see Figure 2). This vein is believed to be an ancient seabed that was pushed upward during the Alps formation.
With sulfate (SO42-) concentrations ranging between 100 to 200 mg l-1 (ca. 2 mM), Lake Cadagno contains up to 10 times more sulfate than most freshwater lakes (Hanselmann and Hutter 1998). Such unusual concentrations of SO4-2
favored the development of sulfate-reducing bacteria (SRB) in the monimolimnion and in the anoxic sediments, thus contributing to an elevated production of hydrosulfide ions (HS-1) that accumulate to levels of 0.5 to 1 mMol in the monimolimnion. In turn, sulfide (H2S) supports the proliferation of a dense community of phototrophic sulfur bacteria in the chemocline at a depth of approximately 12 m where light still reaches the sulfide front that diffuses from the anoxic layer. By metabolizing reduced sulfur compound such as sulfide and thiosulfate (S2O32-
), the phototrophic sulfur bacteria protect the eukaryotic aerobic organisms found in the upper mixolimnion. In fact, phototrophic sulfur bacteria use sulfide as an electron donor for the anoxygenic photosynthesis thus producing elemental sulfur (S0) first and then sulfate. The phylogeny and the population dynamics of phototrophic sulfur bacteria established in the chemocline have been studied since 1993 with
molecular methods based on the sequencing of 16S rRNA genes (Peduzzi et al. 1993; Tonolla et al. 1998a; Tonolla et al. 1999; Tonolla et al. 2005c). The presence of phototrophic sulfur bacteria in Lake Cadagno for as long as 10,000 years ago was confirmed by the analysis of paleo- microbial communities found in the ancient anoxic sediment layers using as molecular tools 16S rRNA genes and quantitative real-time PCR (qPCR) that specifically targeted the contemporary purple and green sulfur bacteria populations (Ravasi et al. 2012).
Figure 2. Bathimetric map of the Lake Cadagno and its hydrogeological context modified from Wirth et al.
(Wirth et al. 2013). Subaquatic springs are shown as white dots and surface in- and out-flows of water are represented by black arrows.
Moreover, Lake Cadagno and its anoxic layer is thought to represent living conditions similar to those found in the Precambrian era (4.5 billion to 545 million years ago) when free oxygen was probably absent from the Earth’s atmosphere. Thus, it may also represent a suitable model for studying the microbial ecology and evolution of life on Earth (Dahl et al. 2010). As meromictic lakes are relatively rare worldwide, Lake Cadagno was the object of numerous studies during the last 30 years leading to the accumulation of reliable data on its chemistry and biology (Peduzzi et al. 1991; Peduzzi et al. 1993; Fischer et al. 1996; Hanselmann and Hutter 1998; Lehmann et al. 1998; Peduzzi et al. 1998; Schanz et al. 1998; Tonolla et al. 1998a; Tonolla et al. 1998b; Tonolla et al. 1999; Bosshard et al. 2000; Lüthy et al. 2000; Camacho et al. 2001;
Peduzzi et al. 2003b; Tonolla et al. 2003; Tonolla et al. 2004; Tonolla et al. 2005c; Tonolla and
Peduzzi 2006; Musat et al. 2008; Decristophoris et al. 2009; Gregersen et al. 2009; Habicht et al.
2011; Storelli et al. 2013a; Wirth et al. 2013).
1.1.2. Biota of the Lake Cadagno
Water stratification resulted in the formation of three distinct habitats throughout the water column of Lake Cadagno. The upper mixolimnion is approximately 12 m deep, and its water is alimented by surface runoff from of a small drainage area of approximately 2 km2 north of the lake that is composed of crystalline rocks of the Gotthard Massif. These crystalline rocks of the watershed are rather resistant to chemical weathering, resulting in a water of the mixolimnion that is poor in salts but rich in oxygen. Waters until a 10 m depth have a low phosphate (PO43-
) content close to the detection limit (<1 μg l-1), with nitrate (NO3-) below 50 μg l-1 and dissolved inorganic carbon (DIC) of approximately 10 mg l-1. Phyto- and zooplanktonic communities are abundant within the first 10 m of the water column, with phytoplankton populations characterized by seasonal changes. In contrast, zooplankton communities are characterized by three major populations found unchanged throughout the year. During the summer, phytoplankton is mostly constituted by vertically and uniformly distributed Pennales and centric diatoms, whereas green algae (Echinocoleum, Sphaerocystis, and Oocystis) dominate during the autumn (Peduzzi et al. 1993; Schanz et al. 1998; Camacho et al. 2001). The chemocline located between the oxygenic mixolimnion and the anoxygenic monimolimnion is approximately 2 m thick and is characterized by steep gradients of light and chemical compounds such as oxygen, sulfide, phosphate, and ammonium (see Figure 1 of the research paper 1). A dense community of anaerobic phototrophic sulfur bacteria of up to 107 cells ml-1 in summer inhabits the chemocline, and includes photosynthetic purple sulfur bacteria (PSB; family Chromatiaceae) of the genera Chromatium, Lamprocystis, Thiocystis and Thiodictyon and green sulfur bacteria (GSB; family Chlorobiaceae) of the genus Chlorobium (Tonolla et al. 1999; Tonolla et al. 2004;
Tonolla et al. 2005c) It was shown that the position of the bacterial layer in the water column is influenced by light conditions and physical displacement of water masses (Wüest 1994; Egli et al. 1998). The food chain connection with the mixolimnion is achieved by zooplankton (Eudiaptomus, Cyclops, Daphnia, Asplanchna) that grazes the bacterial communities established in the chemocline (Schanz and Stalder 1998; Camacho et al. 2001). The monimolimnion that stretches from -13 m to the bottom of the lake (-21 m) is anoxic and rich in reduced compounds because of the absence of oxygen and the activity of anaerobic bacteria, such as SRB. It is characterized by elevated concentrations of sulfate (up to 200 mg l-1), sulfide (≤30 mg l-1), nitrate
(≤5 mg l ), phosphate (≤0.4 mg l ) and DIC (≤6 mg l ). Bacteria are present at high concentrations (up to 105 cells per ml-1) throughout the monimolimnion with Desulfocapsa thiozymogenes (Desulfobulbaceae) and Desulfomonile tiedjei (Syntrophaceae) representing the most abundant species, also in the lower part of the chemocline (Tonolla et al. 1998b; Tonolla et al. 2000; Peduzzi et al. 2003b; Tonolla et al. 2005a; Garrity et al. 2007).
Sediments that cover the lake bottom above the chemocline depth are in contact with oxic water and cover a total area of 474’000 m2. In the southern part of the lake, these sediments are covered by macrophytes (Chara globularis var. globularis and Patamogeton) found between -1 to -7 m. Below this depth, the light and the sulfidogenic character of the bottom support the massive development of phototrophic bacterial mats to a depth of 11 m (Hanselmann and Hutter 1998). Below the chemocline (-12 m) sediments are permanently anoxic, and at a 21 m depth, the upper layer consists mostly of phototrophic sulfur bacteria and algae (mainly diatoms) that settled from the water column with an estimated sedimentation rate of 0.5 cm per year (Birch et al. 1996).
1.2. Phototrophic sulfur bacteria 1.2.1. The sulfur cycle
Sulfur (S) is a brittle, yellow, tasteless, and odorless non-metallic element that is believed to be very abundant in the universe. Most of the Earth's sulfur is present in rocks and salts or buried deep in oceanic sediments, but can also be found in the atmosphere (Brown 1982). The emission of sulfur in the atmosphere comes from natural events such as volcanic eruptions, different bacterial processes (such as decay, respiration or photosynthesis), and evaporation from oceanic water. Furthermore, a large portion of the sulfur emissions in the atmosphere results from human activities, principally air pollution from large industrial areas coming from the combustion of organic fuels. In both cases, sulfur dioxide (SO2) and hydrogen sulfide (H2S) gases are emitted in the atmosphere where they are quickly transformed into sulfate (SO42-
) by reacting with oxygen present in the air. The sulfate is then solubilized by rain water and returns to ground as acid deposition. Plants and microbes assimilate sulfate and convert it into organic forms, introducing it into the food chain. In fact, sulfur is an essential element for life, and it occurs mainly as constituents of protein (cysteine and methionine) but also in various coenzymes (e.g., coenzyme A, biotin, thiamine, etc.). Ultimately, death and subsequent decay of animals and plants reintroduce sulfur into the cycle (see Figure 3A).
Figure 3. (A) The sulfur cycle (1) and its major transformation processes (B) (Tang et al. 2009)
1http://upload.wikimedia.org/wikipedia/commons/4/41/Sulfur_Cycle_%28Ciclo_do_Enxofre%29.png
Of the different numbers oxidation states that exist in nature, sulfur us only found in significant amounts as sulfide (-2), elemental sulfur (0) and sulfate (+6). Transformations between these three major states result from biological as well as chemical processes, although microbial activity plays a primary role and is normally rapid (see Figure 3B).
1.2.1.1. Sulfate reduction. In absence of molecular oxygen, sulfate (SO42-
) is used as a terminal electron acceptor for respiration by dissimilatory sulfate-reducing bacteria (SRB). Sulfate reduction is carried out with a variety of electrons donors such as lactate, pyruvate, malate, acetate, and others, which are oxidized to CO2 at the end of the respiration pathway (Widdel et al. 1992; Hao et al. 1996). SRB can be traced back to 3.5 billion years ago and are considered to be amongst the oldest life forms, having contributed to the sulfur cycle soon after life emerged on Earth (Schinck 1999; Barton and Fauque 2009). Other bacteria such as Proteus, Campylobacter, Pseudomonas and Salmonella also possess the ability to reduce sulfur but as facultative anaerobes they can use oxygen or other terminal electron acceptors. Normally, sulfide produced during anaerobic respiration is released into the environment thus contributing to maintain anaerobic conditions. Moreover, many bacteria can reduce small amounts of oxidized sulfur for the biosynthesis of cysteine, methionine, and other cofactors. The incorporated inorganic sulfate is first transformed in adenosine-5’-phosphosulfate (APS) by ATP sulfurylase and then further converted into 3′-phosphoadenosine-5′-phosphosulfate (PAPS) by the APS kinase, with PAPS playing a key role in sulfate transfer reactions to numerous substrates (Lin et al. 1995; Leustek et al. 2000; Negishi et al. 2001; Saito 2004).
1.2.1.2. Sulfide oxidation. Hydrogen sulfide (H2S), that is the most reduced form of inorganic sulfur, results from the activity of SRB found in sediments or in hydrothermal vents (see above 1.2.1.1. Sulfate reduction). Although sulfide is toxic to most organisms, mainly because of it interferes with aerobic respiration (Reiffenstein et al. 1992), it serves as electron donor for the energy-generating systems of photo- and chemo-lithotrophic bacteria as well as some Archaea (Brune 1995a; Van Gemerden and Mas 2004). During the autotrophic growth of these bacteria, sulfide provides electrons to a membrane-bound electron transport system in a process that ultimately leads to the production of reducing power such as NAD(P)H and reduced ferredoxin, which are required for fixation of the inorganic carbon. This transfer of electrons requires energy that is provided by light in phototrophic bacteria (Blankenship and Hartman 1998) and by dark redox reactions of inorganic substrates in chemo-lithotrophic bacteria (Kelly 1999).
1.2.1.3. Sulfur disproportionation. Sulfur disproportionation (dismutation or inorganic fermentation) is an energy-yielding process in the metabolism of numerous genera of SRB, which use inorganic sulfur compounds of intermediary oxidation states (elementary sulfur S0, thiosulfate S2O32-
and sulfite SO32-
) as both donors and acceptors of electrons (Bak and Cypionka 1987; Cypionka et al. 1998). This metabolic process involved in sulfur transformation was shown to be particularly important in aquatic systems (Jörgensen 1990; Jörgensen et al. 1991).
1.2.2. Ecological distribution of phototrophic sulfur bacteria
Phototrophic sulfur bacteria are organisms commonly found in illuminated anaerobic aquatic environment containing hydrogen sulfide. In particular, these bacteria favour (1) stratified lakes where they occupy the top of the anoxic layer, and (2) the top layers of anoxic illuminated sediments on which they often establish colored microbial mats (Van Gemerden and Mas 2004).
In holomictic lakes, where a thermal stratification is established transiently during summer, phototrophic sulfur bacteria are present as seasonal communities, whereas the permanent water stratification in meromictic lakes allows for the formation of stable communities throughout the year. A number of physico-chemical parameters influence the development of specific phototrophic sulfur microorganisms including the intensity and quality of light, which is affected by planktonic organisms (algae, bacteria) found in the water column, as well as the depth and chemistry of the chemocline (Abella et al. 1980; Vila and Abella 2001). Microbial mats are stratified environments that share some of the structural features of meromictic lakes, but on a smaller scale. Although these mats often harbor bacterial communities that differ significantly in composition and stability, the development of phototrophic sulfur bacteria is usually restricted to the upper 5 mm due to the rapid extinction of transmitted light into sediments. In aquatic planktonic systems, the infrared and UV part of the light spectrum are rapidly attenuated because of absorption and scattering, whereas in sediments, infrared radiation appears to penetrate deeper (Parkin and Brock 1980; Jorgensen and Des Marais 1986; Veldhuis and van Gemerden 1986).
Phototrophic organisms use a number of pigments (e.g. carotenoids) to capture light energy. In the case of purple sulfur bacteria (PSB), the most abundant pigment is okenone while spirilloxanthin, lycopenal and rhodopinal are clearly less abundant. In the case of green sulfur bacteria (GSB) pigments such as chlorobactene (green-colored species) and isorenieratene (brown-colored species) are normally observed. Carotenoids are not the only class of pigments
found in phototrophic sulfur bacteria: the bacteriochlorophylls (BChl), in particular forms a and b for PSB and form c, d and e for GSB, were found to be essential for absorbing light and allowing a photoautotrophic growth. Pigments preserved in anoxic sediments or detected in living organisms are also used as markers to study a number of environmental conditions, evolutionary processes and biological origins (Xiong et al. 2000; Brocks and Schaeffer 2007).
1.2.3. Photosynthetic inorganic carbon fixation in PSB and GSB
Phototrophic sulfur bacteria are considered as anaerobic organisms transducing light energy into a biologically useful form without the generation of oxygen from the oxidation of water (they lack photosystem II). Instead of oxidizing water, these microorganisms use reduced forms of sulfur or possibly hydrogen gas (H2) as electrons donor for the reduction of CO2. The energy necessary for the process of CO2 fixation is generally provided by light, which is very efficiently collected by light-harvesting antenna pigments and then transferred to the photosynthetic reaction center, where electrons are displaced to specific acceptors such as NAD(P)+ and ferredoxin, creating at the same time a transmembrane potential (proton-motive force) used for the production of ATP (Overmann and Garcia-Pichel 2006).
Phototrophic sulfur bacteria are essentially divided into two major groups: the purple sulfur bacteria (PSB) and the green sulfur bacteria (GSB) (Imhoff 2004). In general, the GSB are considered to be obligate photo-autotrophs, whereas PSB are capable of both photo-autotrophy and photo-heterotrophy (Parkin and Brock 1981). GSB and PSB are also known to display different sensitivities towards molecular oxygen (O2), with GSB being obligate phototrophic anaerobes whereas some species of PSB that can may grow chemotropically in the presence of molecular oxygen, mainly under microaerophilic conditions (Trüper 1981; Kämpf and Pfennig 1986; de Wit and van Gemerden 1987). Three major structural and/or metabolic differences distinguish PSB from GSB: (1) the pigment composition (BChl and carotenoids) that affects the structure of the light-harvesting antenna and a different type of photosynthetic reaction center, (2) the CO2 fixation pathway, and (3) the deposition of sulfur globules inside of PSB or outside of GSB the cells.
1.2.3.1. Pigments, light-harvesting antenna and photosynthetic reaction center. As stated above, different sets of pigments are observed in the PSB and GSB, in particular, the molecules of bacterio-chlorophyll (BChl) composing the light-harvesting antenna differentiate PSB from
GSB. As described above, the dominant pigments found in PSB are BChl a and b, while in GSB BChl c, d and e are the most abundant pigments. BChl a also occurs in GSB, but in lower quantities in photosynthetic reaction centers and not in antenna. PSB and GSB also differ in terms of the structural properties of their light-harvesting complexes. In GSB, these complexes are organized into highly structured organelles known as chlorosomes (Frigaard and Bryant 2006), allowing an highly efficient absorption of light energy. In fact, GSB appear to be better adapted than PSB to grow at low irradiance intensities, and thus are normally found deeper or just under the layer of PSB when coexisting in the same habitat. Two light-harvesting complexes (LH1 and LH2) are observed in PSB, and LH1 consists in pairs of small transmembrane polypeptides to which BChl is non-covalently attached in a 1:1 stoichiometry, forming with the reaction center a RC-LH1 ‘core’ complex. In contrast, the LH2 (or peripheral) antenna complex is only present in some purple bacteria (Law et al. 2004). The role of pigments in the light- harvesting antenna is to capture and then transfer the energy of the light to the photosynthetic reaction center. Photosynthetic reaction centers are divided into two main groups: type I, in which a ferredoxin (Fe-S) cluster acts as the terminal electron acceptor generally in GSB, and type II which in PSB and others phototrophic bacteria use a quinone as a terminal electron acceptor. Ultimately, the transit of the electrons from the photosynthetic reaction center to the cytochrome complex and the NAD(P)+ or other cofactors (e.g. ferredoxin in GSB), establishes a proton-motive force across the membrane, which is finally used to produce ATP by ATPases.
1.2.3.2. Pathway for fixing CO2. Although PSB and GSB use the reduced cofactors and ATP accumulated during photosynthesis, pathways involved in fixing CO2 are distinct (Sirevag et al.
1977; Berg 2011). PSB normally assimilate CO2 using the Calvin-Benson-Bassham cycle (CBB cycle) (see Figure 4A) in which the key enzyme is the ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO). In contrast, GSB preferentially use the reductive reverse tricarboxylic acid (rTCA) cycle (see Figure 4B).
The CBB cycle can be divided into three major steps: (1) carbon fixation, (2) reduction of the fixed carbon, and (3) regeneration of molecules involved in the cycle. The first key reaction of carbon fixation is performed by RuBisCO that catalyzes the carboxylation of ribulose-1,5- bisphosphate (RuBP) into two molecules of 3-phosphoglyceric acid (3-PGA) using CO2 as substrate. Then 3-PGA is further metabolized and reduced using ATP and NAD(P)H in triose phosphate. The CBB cycle produces one molecule of triose phosphate (glyceraldehyde-3- phosphate, GAP) from 3 molecules of CO2. Ultimately the cycle regenerates RuBP as an
acceptor for CO2. These reactions of regeneration in the CBB cycle are essentially catalyzed by non-specific enzymes also involved in others metabolic pathways, except for the phosphoribulokinase that regenerates RuBP from ribulose-5-phosphate (Ru-5-P) using one molecule of ATP. In total, the CBB cycle consumes nine ATP and six NADPH equivalents for the synthesis of one triose phosphate molecule.
The citric acid (TCA) cycle, also known as “Krebs cycle”, is a process utilized by aerobic organisms to generate energy (ATP) and reducing agents (NADPH) via the oxidation of acetate that is derived from carbohydrates, fats, and proteins into CO2. However, some microorganisms such as GSB favor the reverse reactions of the TCA (thus called rTCA cycle) in order to fix CO2
in triose phosphate. Several enzymes required for the rTCA are also active in the Krebs cycle, but a number of irreversible reactions need specific enzymes such as a fumarate reductase, one ferredoxin-dependent 2-oxoglutarate synthase, and an ATP-citrate lyase. These three enzymes are usually regarded as the characteristic enzymes of the rTCA cycle. As electron donors, the rTCA cycle uses reduced ferredoxin and NAD(P)H, which explains the presence in GSB of type I-photosynthetic reaction centers capable of reducing ferredoxin. Compared to the CBB cycle, the rTCA cycle seems more energy efficient since it requires only five ATPs to convert CO2 to triose phosphate.
Figure 4. Metabolic pathways for assimilating inorganic carbon: (A) the Calvin-Benson-Bassham cycle (CBB cycle) active in PSB, while GSB use the (B) reductive reverse tricarboxylic acid (rTCA) cycle (Berg 2011). The characteristic enzymes for each pathway are underscore.
(A) Enzymes: 1, RuBisCO; 2, 3-phosphoglycerate kinase; 3, glyceraldehyde-3-phosphate dehydrogenase; 4, triose-phosphate isomerase; 5, fructose-bisphosphate aldolase; 6, fructose-bisphosphate phosphatase; 7, transketolase; 8, sedoheptulose- bisphosphate aldolase; 9, sedoheptulose-bisphosphate phosphatase; 10, ribose- phosphate isomerase; 11, ribulose-phosphate epimerase; and 12, phosphoribulokinase.
(B) Enzymes: 1, ATP-citrate lyase; 2, malate dehydrogenase; 3, fumarate hydratase; 4, fumarate reductase; 5, succinyl-CoA synthetase; 6, ferredoxin-dependent 2-oxoglutarate synthase; 7, isocitrate dehydrogenase; 8, aconitate hydratase; 9, ferredoxin-dependent pyruvate synthase; 10, pyruvate/phosphoenolpyruvate synthase; 11, pyruvate/phosphoenolpyruvate carboxylase.
1.2.3.3. Sulfur globules. The ability to store surplus of various substances during unbalanced growth is a widespread strategy amongst microorganisms that need to cope with fluctuating environments. Phototrophic sulfur bacteria using reduced sulfur compounds during anoxygenic photosynthesis can accumulate sulfur globules, which are further oxidized to sulfate when the pool of reduced sulfur compounds becomes exhausted. In addition, it was shown that in the dark stored sulfur could be reduced to sulfide during the oxidation of glycogen because of the excess of electrons produced during the anaerobic breakdown of glucose (Van Gemerden 1968;
Paschinger et al. 1974; Trüper 1978). Sulfur globules occur extracellularly in GSB as well as in the PSB members of the family Ectothiorhodospiraceae, while they are stored intracellularly in other PSB members of the Chromatiaceae (Frigaard and Dahl 2008). Irrespectively of their
storage site, these globules consist of long sulfur chains generally terminated by organic residues similar to glutathione, most likely responsible for keeping the sulfur in a “liquid” state at ambient pressure and temperature conditions (Steudel 1996; Prange et al. 2002; Prange and Modrow 2002). Interestingly, whole-cell flotation experiments indicated that sulfur inside globules had an unexpectedly low density of approximately 1.2, compared with the common density of elemental sulfur (Guerrero et al. 1984; van Gemerden et al. 1985). This suggests a possible hydration of the sulfur to long hydrophilic chains once inside globules.
Intracellular sulfur globules by the PSB members of the Chromatiaceae appear in most cases to be separated from the cytoplasm by a unit membrane which may be continuous with the cytoplasmic membrane, depending on the organism (Pattaragulwanit et al. 1998). For example, in Allochromatium warmigii sulfur globules appeared as located at the two cell poles, whereas in Lamprocystis and Thiodictyon in the peripheral part of the cell and in Lamprobacter modestohalophilus in the “center” of the cell. Oxidation of sulfide at the outer surface of the cytoplasmic membrane, adjacent to the sulfur inclusions, may establish a proton gradient necessary for ATP synthesis and reduce the potential for sulfide toxicity within the cytoplasm.
Sulfur globules are delimited by a protein envelope composed of 2 major types of “sulfur globule proteins” (SGP) of ca. 10.5 kDa (SgpA and SgpB) and 8.5 kDa (SgpC) (Brune 1995b;
Pattaragulwanit et al. 1998; Prange et al. 2004; George et al. 2008).
In Ectothiorhodospira and GSB organisms, sulfur globules appear to be attached to the outer membrane of the cell wall via both spinae and capsules (Rojas et al. 1995; Pibernat and Abella 1996), thus making them difficult to be acquired by competing bacteria (van Gemerden 1986).
Microscopic observations indeed confirmed that most sulfur compounds remained attached to cells, with little being free to float in the medium (Trüper 1984).
1.2.4. Phototrophic sulfur bacteria in the Lake Cadagno
The crenogenic meromictic Lake Cadagno possesses all of the characteristics needed to support sulfate-reducing and phototrophic sulfur bacteria. The permanent water stratification and the abundance of sulfur compounds brought by sublacustrine springs, allowed the formation of an environment favorable to anoxygenic sulfur bacteria (Pfennig 1975). The abundance of phototrophic sulfur bacteria is especially pronounced in the upper part of the anaerobic water layer, at the level of the chemocline, where a dense population can generally be detected, especially during the summer (Schanz et al. 1998; Del Don et al. 2001).
During the past 20 years, the major populations of phototrophic sulfur bacteria established in the chemocline and their role in the global economy of the lake were investigated (Peduzzi et al.
1993; Fischer et al. 1996; Tonolla et al. 1998b; Bosshard et al. 2000; Lüthy et al. 2000; Tonolla et al. 2000; Camacho et al. 2001; Peduzzi et al. 2003a; Tonolla et al. 2003; Tonolla et al. 2005b;
Musat et al. 2008; Habicht et al. 2011). In particular, Tonolla et al. highlighted the presence of several new taxa of PSB (δ-Protobacteira - Chromatiaceae) and GSB (Chlorobi - Chlorobiaceae) (see Figure 5) (Tonolla et al. 1998a; Tonolla et al. 1999; Tonolla et al. 2004). Long-term monitoring of the phototrophic sulfur bacteria in the chemocline of Lake Cadagno also indicated the prevalence until 2002 of two major PSB populations: Chomatium okenii and Candidatus
“Thiodictyon syntrophicum,”, the later of which was initially identified as Lamprocystis sp.
population F. After the year 2000, GSBs initially represented only by Chlorobium pheobacteroides at low densities became preponderant, due to the unusual development of another new species C. clathratiforme. Such major change in community structure inside the chemocline was also accompanied by changes in the turbidity, sulfide concentration and light profiles. These changes were proposed to result from strong climatic events, such as autumnal windstorms that caused strong mixing events and a temporary disruption of the chemocline. The appearance of C. clathratiforme caused a PSB to GSB shift in dominating populations of chemocline bacteria, as well as a rise in the total number of phototrophic sulfur bacteria from ca.
106 to 107 cells per ml-1. Currently, C. clathratiforme is estimated to represent up to 95% of the phototrophic sulfur bacteria present in Lake Cadagno (Tonolla et al. 2005c; Decristophoris et al.
2009; Gregersen et al. 2009).
Figure 5. (A) Neighbour-joining phylogenetic tree of DNA sequences of selected clones from a 16S rRNA gene library of microorganisms found in the chemocline of Lake Cadagno, together with published sequences archived in the the EMBL and GenBank databases. The distance scale indicates the expected number of changes per sequence position. Pictures from FISH experiments of the major populations of phototrophic sulfur bacteria are showed beside the corresponding clones (Tonolla et al. 2005c).
By using a series of agar-shake dilutions as in Pfenning (1978) in combination with various molecular techniques, the main populations of phototrophic sulfur bacteria could be isolated and grown separately in liquid media. Currently and with the exception of Ch. okenii, all main populations of phototrophic sulfur bacteria living in the chemocline of Lake Cadagno are being kept as pure cultures in our laboratory (see Figure 5). Among these populations, three were described as new species: Thiocystis cadagnonensis, T. chemoclinalis (Peduzzi et al. 2011), and the Candidatus “Thiodictyon syntrophicum” strain Cad16T, which has also the ability to form aggregates with the SRB Desulfocapsa thiozymogenes (Peduzzi et al. 2003a; Peduzzi et al.
2012).
The aforementioned previous studies on the diversity and dynamics of the population of phototrophic sulfur bacteria in the chemocline of Lake Cadagno represent an important starting point for subsequent functional and ecological studies. By fixing inorganic carbon using reductants (NAD[P]H) and energy (ATP) generated from sulfide oxidation (see paragraph 1.2.3), phototrophic sulfur bacteria prevent diffusion of toxic sulfide into the oxygen-rich mixolimnion in which fish, algae, phyto- and zooplankton as well as other aerobic bacteria thrive (Lüthy et al.
2000; Hell et al. 2008). Studies of the primary production in the lake showed that approximately half of the total CO2 assimilation of the lake occurred in the small volume of the chemocline (Camacho et al. 2001). Moreover, the rates of dark CO2 fixation in the chemocline were even higher than rates of photo-assimilation, especially at depths where the oxygen and sulfide coexisted during part of the day (Camacho et al. 2001).
