Conclusions and perspectives

The metabolism of phototrophic sulfur bacteria still remain poorly understood. Anoxygenic phototrophic sulfur bacteria occupy regions where light reaches anoxic layers in water columns or in sediments. The mechanism of CO₂ fixation in the presence of light by the Calvin-Benson-Bassham cycle (present in PSB) or the reverse tricarboxylic acid cycle (present in GSB) is relatively well characterized, while the assimilation of inorganic carbon in the dark remains poorly understood. However, previous studies have confirmed the pronounced capacity of PSB to fix CO2 in the dark (Wood and Stjernholm 1962; Cohen et al. 1977; Camacho et al. 2001;

García-Cantizano et al. 2005; Casamayor et al. 2008; Casamayor 2010; Casamayor et al. 2012).

This study aimed primarily at determining the ecological role of the phototrophic sulfur bacteria in the primary production of the Lake Cadagno. Previous reports showed that despite a volume of only 10% of Lake Cadagno, the chemocline is responsible for up to 40% of the total inorganic carbon photo-assimilation, and that significant rates of CO2 assimilation occurred during the night, indicating that primary production also relies on mechanisms other than photosynthesis (Camacho et al. 2001). In Chapter 2, we confirmed that carbon assimilation occurred at the rate of approximately 297 ng of 14C mL^-1 in the light and approximately 231 ng of 14C mL^-1 in the dark. Amongst the four major population living in the chemocline tested during the initial in situ experiment, the dominant small-celled species Candidatus “Thiodictyon syntrophicum” strain Cad16^T was estimated to be responsible for up to 25% of the total primary production in the chemocline. Following a detailed taxonomic characterization, Cad16^T was proposed as the type strain of a new species belonging to the genus Thiodictyon (see Chapter 3).

Laboratory experiments using pure cultures of strain Cad16^T grown in autotrophic media showed that the maximal CO₂ assimilation rate occurred during the first 4 h of light (07:00 to 11:00 AM). Furthermore, mRNA analyses confirmed that the two genes in the draft genome of strain Cad16^T coding for RuBisCO forms I (CbbL) and II (CbbM) were differentially expressed. While cbbM was constitutively expressed at a basal level throughout light and dark periods, the expression of cbbL varied during the light-dark cycle and was modulated by the available carbon sources. Yet, peaks in cbbL expression did not correlate with the periods of maximal CO₂ assimilation.

Using a proteomic approach (Chapter 4), we were able to show the presence of three proteins that were up-regulated in the dark and may be involved in the anaerobic dicarboxylate/4-hydroxybutyrate (DC/HB) cycle described until now only in Archaea populations (Huber et al.

2008; Berg 2011). However, these three enzymes are also part of two other pathways: the

(r)TCA cycle (malate dehydrogenase) and the PHB degradation pathway (3-ketoacyl-ACP reductase and acetoacetyl-CoA-ketothiolase). Moreover, strain Cad16^T could hypothetically fix CO2 in the dark via the CBB cycle. As shown in other studies, when the two different forms of the enzyme RuBisCO are present, RuBisCO form II is typically involved in maintaining the redox balance when the cells have an excess of reducing power (Dubbs and Robert Tabita 2004;

Joshi et al. 2009; Laguna et al. 2010). Both hypotheses need to be tested by additional analyses, such as quantification of key genes by qRT-PCR and other proteomics assays. Nevertheless, the proteomic approach (2D-DIGE) performed in this study using a phototrophic sulfur bacterium has important biological relevance because it provides concrete indications of the mechanism involved in the process of CO2 assimilation in the dark. In the presence of light, PSB accumulate storage compounds such as intracellular sulfur globules, polyhydroxybutyrate (PHB) granules, polyphosphate and glycogen (Del Don et al. 1994; Van Gemerden and Mas 2004). Proteomic data confirmed the presence of enzymes potentially involved in the production of PHB, the most common polyhydroxyalkanoate (PHA). PHB that serves as a carbon and energy reserved, also represents a sink for reducing equivalents. We propose that the energy and reducing power stored in the PHB granules play a key role in the process of dark CO₂ fixation. This suggests that PSB are capable of CO₂ fixation in the dark because they accumulated sufficient reserves during the light period. Moreover, PHB is a biotechnologically interesting polymer since it is biodegradable and derived from renewable resources. PHAs in general have attracted much interest and have been used in the development of many technical and medical applications in recent years (Chen 2009; Keshavarz and Roy 2010). Currently, commercial products (e.g., Biopol^®, manufactured by Zeneca BioProducts) are synthesized by glucose fermentation using Ralstonia eutropha H16 mutants (Asrar and Gruys 2002). The possibility of producing this commercial product using light instead of glucose could be an interesting future application.

Another interesting topic for future investigation is the characterization of hypothetical carboxysome-like microcompartments in which form I RuBisCO (CbbL-CbbS) may accumulate and thus enhance CO2 fixation. All of the genes required for the formation of these microcompartments are present in the draft genome of strain Cad16^T. As in other microorganisms, the operon containing cbbL (JQ693373) also includes cbbS (JQ693374), as well as six genes putatively coding for components of a carboxysome-like microcompartment (JQ693375-JQ693380) (Cannon et al. 2010).

In conclusion, our analyses showed that while GSB represent approximately 95% of the total population of phototrophic sulfur bacteria, it is the minor fraction (ca. 5%) of PSB that carry out most of the CO2 fixation in the chemocline of Lake Cadagno. Amongst PSB, it appears that the newly characterized type strain Cad16^T which accounts for approximately 30% of the total PSB population, is the most active CO2 fixing microorganism regardless of the presence of absence of light. Surprisingly, transcription patterns of the RuBisCo encoding genes cbbL and cbbM did not correlate with maximal levels of CO2 fixation indicating that primary production by Cad16^T involved more complex mechanisms than just presence of RuBisCO. For example, presence of carboxysomes need to be further explored. In addition, CO2 fixation in absence of light may require reserve polymers, such as PHB, that are accumulated during the day and will provide enough energy and reducing power during the night. The DC/HB and/or the CBB cycles were identified as candidate pathways for the dark CO2 fixation process. This study represents a strong starting point for future analyses to further explore the dark CO₂ fixation observed in the model PSB strain Cad16^T.