Short-term dynamics of Bdellovibrio and like organisms in Lake Geneva in response to a simulated climatic extreme event

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Habitats, abundance and distribution of BALOs

In certain occasions, the abundance of BALOs can reach a substantial percentage of the bacterial populations. However, in most cases, BALOs are characterized by low abundances, whether detected by culture-dependent methods or DNA surveys, with the latter is able to recover a larger number of strains. Overall, they do not form dominant populations (Shemesh et al., 2003) unless prey are abundant and are in a closed system (Kandel et al., 2014). For example, the study of marine BALOs is more amenable in estuarine systems rather than in the open ocean (Williams and Piñeiro, 2006). In fact, when using specific primers for the detection of BALOs, BALOs reads and OTUs (operational taxonomic units) were less frequent in open water, i.e., Banyuls Bay (France) than in peri-alpine lakes (Geneva and Annecy), which constitute smaller and relatively closed systems (Ezzedine et al., 2020a). In addition, the abundance of BALOs which may vary between a few cells to >104 cells per mL, is affected by the quality of the environment. For instance, polluted clouds (Amato et al., 2017) and polluted river water (Staples and Fry, 1973) are richer in BALOs than their unpolluted counterparts. Other polluted environments, such as sewage, are also rich in BALOs (Staples and Fry, 1973). This is likely due to the presence of a greater number of potential prey. In such environments, BALOs are readily detectable and therefore the probability of isolating them is higher. However, their abundance has not often been quantified. Only Zheng et al. (2008), Kandel et al. (2014), Van Essche et al. (2009) and Iebba et al. (2013) reported abundances using quantitative PCR (qPCR). For example, the count of Halobacteriovorax in Baltimore Harbor reached 8,570 copies per mL (Zheng et al., 2008), and for Bdellovibrio in the human gut; 2 to 549 copies per mg (Iebba et al., 2013). These reports have been slightly supplemented with data obtained for peri-alpine lakes such as for Lake Geneva where minimum and maximum values were measured to vary between 38 to 1,253 and 5 to 37,270 copies per mL for Bdellovibrio and Peredibacter respectively (Figure 4) (Ezzedine et al., 2021). In general, the BALO group have great adaptability to different environments (Yu et al., 2017) and they are therefore detected in many places. In natural and anthropic habitats they can be found as free living cells (planktonic), bdelloplast or those associated with biofilm (Markelova and Gariev, 2005; Jurkevitch, 2012a). Furthermore, HI variants, such as B. bacteriovorus, are seen in laboratories to form tenacious biofilms on abiotic surfaces (Medina and Kadouri, 2009). To date, BALOs have been reported in soils, rhizosphere plant systems, rivers, lakes, seas, oceans, estuaries, sediments, coral reefs, salty-ponds, mangroves, Antarctica, geothermal waters, oceanic subsoil, anoxic environments, shrimp aquaculture basins, leeches, activated sludge, waste waters, human and animal intestines, human lungs, blue crab gills, feces, and polluted and unpolluted clouds (Sutton and Besant, 1994; Schwudke et al., 2001; Davidov and Jurkevitch, 2004; Rendulic et al., 2004; Williams and Piñeiro, 2006; Davidov et al., 2006a; Wen et al., 2009; Kikuchi et al., 2009; Van Essche et al., 2011; Cao et al., 2015; Williams et al., 2016, 2018; Aguirre et al., 2017; Amato et al., 2017; Paix et al., 2019; Ezzedine et al., 2020b) and more recently in forest leaves (Miura et al., 2019) (Fig. 4). Their presence in so many environments demonstrates the widespread presence of BALOs, but not all strains (as for any bacterium) are found in every type of ecosystem. Many factors influence the distribution of BALOs and their diversity. For example, a salinity above 0.5% is required to detect Halobacteriovorax species. Moreover, among the saltwater BALOs, subpopulations are adapted to either low, moderate or extreme salinities (Amat and Torrella, 1989; Williams and Piñeiro, 2006). Halobacteriovorax were never reported in freshwater; however, Bdellovibrio, Peredibacter, Bacteriovorax and Micavibrio DNA are found in fresh and salt waters as shown in our previous studies (Ezzedine et al., 2020a, 2020b). Moreover, qPCR results show that Peredibacter is clearly more abundant than Bdellovibrio and Bacteriovorax and is predominant in the epilimnion of Lake Geneva and the metalimnion of Lake Annecy (Ezzedine et al., 2021). Culture-based approaches or RNA surveys are required to further confirm the presence of active BALOs in these ecosystems. Kandel et al. (2014) also reported the presence of freshwater BALOs (Bdellovibrio and Bacteriovorax) in a saline mechanical zero discharge system. The presence and abundance of BALOs should only be attributed to active and living individuals. For example, when using a culture-based method, BALOs were reported to be more concentrated in the air-water surface microlayer than below that surface (Williams, 1987). In another study (Ezzedine et al., 2020b) using a DNA survey, the DNA of BALOs were detected at a greater abundance at 200 m than at the surface. These differences are probably due to DNA originating from dead BALOs that may sedimented in the depths, but it cannot be ruled out that active BALOs could be present in these places. Other factors also influence the distribution of BALOs such as oxygen availability and temperature (Schoeffield et al., 1996; Shemesh et al., 2003; Kandel et al., 2014; Williams and Chen, 2020). BALOs are aerobic, but halotolerant strains are reported to tolerate microaerobic conditions and survive anoxic periods as attack phase cells or as bdelloplast (Schoeffield et al., 1996). Moreover, BALOs exhibit seasonal distribution and some BALOs are more abundant or detectable in certain months (Williams et al., 1982; Ezzedine et al., 2020b). In fact, warmer months (late summer and early fall months) were reported to allow a greater recovery of marine BALOs. In winter, marine BALOs were recovered in higher abundance from the sediment (< 5 cm (Fry and Staples, 1976)) than in the water column (Williams et al., 1982). Thus, Williams & Piñeiro (2006) stipulated that BALOs in sediment are similar to “seeds” that allow BALOs to thrive again. Overall, BALOs are reported to be more abundant in sediments and biofilms than in the water column (Williams et al., 1982, 1995). Biofilms contain myriad of bacteria, including prey bacteria, and offer protection from environmental conditions; therefore, BALOs most likely thrive the best in such habitats (Williams et al., 1995; Kelley et al., 1997; Markelova and Gariev, 2005). This is in accordance with Markelova (2002), who suggested that BALOs survive better in harsh environmental conditions (e.g., polluted areas) when they are surface associated compared and more likely as bdelloplast with an arrested stage growth.

Biotechnological applications of BALOs

Like bacteriophages used in phage therapy (Górski et al., 2018), BALOs are found in a variety of environment and are probably ingested safely and unconsciously on a daily basis (Willis et al., 2016). Their predation ability makes them also candidates for many applications dealing with the biological control of some bacterial populations (Sockett and Lambert, 2004; Harini et al., 2013; Cao et al., 2015; Bratanis et al., 2020). In addition, their predation skills have been verified on more than 100 human pathogens (Mun et al., 2017), such as E. coli, Salmonella and Klebsiella pneumonia (Kadouri et al., 2013; Shatzkes et al., 2016). Gram-negative bacteria are typically responsible for more than 30% of nosocomial infections (Hidron et al., 2008; Baker et al., 2017), and they are often associated with very high levels of morbidity and mortality in intensive care units (Gaynes and Edwards, 2005; Baker et al., 2017) due to bacteria that are multiresistant to antibiotics (Kadouri et al., 2013; Monnappa et al., 2014). Therefore, the use of alternative solutions to control such infections has become a necessity. BALOs have been suggested as a replacement of antibiotics to combat multiresistant pathogens in human and other organisms (Sockett and Lambert, 2004; Johnke et al., 2017b) or as a complement of antibiotic treatments since the genomes of epibiotic and endobiotic BALOs present a large number of antibiotic resistance genes (Pasternak et al., 2014). For example, Marine et al. suggested employing B. bacteriovorus with antibiotics, such as trimethoprim, for cotherapy (Marine et al., 2020). Additionally, according to Hobley et al. (2020), B. bacteriovorus can be combined with bacteriophages to increase the eradication of E. coli. Furthermore, the large number of proteases and other hydrolases in BALOs constitutes a valuable reservoir of enzyme-based antimicrobial substances (Rendulic et al., 2004). Several groups of researchers, including Im et al. (2017), have suggested that these predatory bacteria are not harmful to human and animal cell cultures. In addition, the predatory bacteria inoculated in animal models, such as mice, rats, rabbits, guinea pigs or hens, show no toxicity (Verklova, 1973; Westergaard and Kramer, 1977; Atterbury et al., 2011; Dwidar et al., 2012; Shatzkes et al., 2016). To date, no diseases have been associated or attributed with any BALO infection (Willis et al., 2016). In fact, BALOs such as B. bacteriovorus carry out a highly orchestrated and targeted hydrolytic attack on prey bacteria due to type I and II secretion systems and from the protein export system: “Twin arginine translocation” (Tat) (Wang et al., 2011; Rotem et al., 2014). Moreover, the lipopolysaccharide membrane (LPS) of B. bacteriovorus is neutral and thus they are weakly immunogenic for humans or animals (Willis et al., 2016). In other words, there is an absence of a strong and sustained inflammatory response in the presence of predatory bacteria, unlike other bacteria, which have negatively charged LPS. Additionally, predators have coevolved alongside prey bacteria and thus encode various predatory enzymes that are difficult to counter with a simple mutation (Negus et al., 2017). Only reversible phenotypic resistance to the predation of BALOs has been previously reported (Shemesh and Jurkevitch, 2004). This resistance probably originates from the release of enzymes from the prey and predator that causes the prey to harden its cell wall or from the accumulation of predation waste that can act as a physical barrier to predator attachment (Shemesh and Jurkevitch, 2004). However, in an in vivo circulatory system, an accumulation of waste products that can hinder attachment is not likely to occur (Enos et al., 2017). Furthermore, most BALOs do not replicate outside of the prey and therefore they express few transport proteins and few surface epitopes for recognition by the prey’s immune system (Gupta et al., 2016; Monnappa et al., 2016; Negus et al., 2017). Another advantage of their use as antimicrobial agents is that BALOs invade Gram-negative pathogens without using a receptor-based recognition system, which makes it difficult for prey to acquire any genetic resistance (Willis et al., 2016). Finally, multiple strains of BALOs exist and therefore therapies using different species of BALOs to reduce resistance are easily conceivable such as phage cocktails promoted in phage therapy (Chan et al., 2013).

Understanding pathogen predation of BALOs in vivo

To use BALOs as a biocontrol agent in the near future, on one hand it is essential to characterize if pathogenic prey exhibit predation-resistant phenotypes. On the other hand, it is also necessary to establish a list of prey for each predator since each BALO strain has a different predation spectrum. The evolutionary dynamics of the evolution of the predator genome can be modeled or monitored over the long term to ensure that the BALO strain does not become pathogenic for eukaryotic cells (Enos et al., 2017). However, for an actual application in a human or nonhuman host, it is necessary to go beyond tightly controlled prey-predator systems in conventional laboratory buffer solutions. For example, the presence of other bacterial species, which act as a disruptor, must be considered when evaluating the clinical application of BALOs against simple and mixed infections. Natural environments are very diverse and complex. Moreover, in the human body, several immunological and antimicrobial factors, such as antibodies, antimicrobial peptides and leukocytes, can act on predatory bacteria. These factors can disrupt or cause the death of bacterial predators before they begin their predation cycles. For example, Baker et al. (2017) observed that B. bacteriovorus predation on K. pneumoniae was delayed since the predators shape changed from vibroid to round when exposed to human serum. However, this form is reversible after acclimation of the predators. In addition, two solutions were offered to avoid such deformity: pre-exposing the predators to human serum or developing resistant strains to human serum by directed evolution over several generations. Additionally, Im et al. (2017) found that the albumin of the human serum inhibits predation by completely coating predatory cells, preventing in fine the predator from attacking its prey. These same authors reported that some antibodies in human serum recognize certain BALOs strains or their surface constituents. They also specified that the osmolality of blood serum (285 – 295 mOsm kg-1) severely inhibits nontolerant BALOs strains. Fortunately, there are numerous BALOs strains that are present in a multitude of habitats, including halotolerant BALOs, such as Halobacteriovorax. This strain hypothetically could be considered for human serum instead of B. bacteriovorus (further experimentation are needed). Another problem was noticed in vivo where the population of pathogenic bacteria, which was initially reduced by the predation of B. bacteriovorus, succeeded in re-emerging after a few hours. This suggests the development of resistance by pathogens and this resumption of growth can jeopardize the use of BALOs as biotherapeutic agents (Baker et al., 2017). However, the predator-pathogen interaction has been shown to display different outcomes according to ecological conditions (Gallet et al., 2009). To explain the emergence of resistant prey, it is necessary to invoke the hypothesis stating that the presence of cellular debris from predation can disturb the course of predation. Debris can be consumed by prey for its metabolism and therefore promote a resumption of its growth. In addition, debris that are not used by the prey and therefore remain in the environment can contribute to the emergence of a resistance phenotype (Shemesh and Jurkevitch, 2004; Baker et al., 2017). In addition, the mode of administration of the predator is essential to guarantee the success of eliminating pathogens. Indeed, Shatzkes et al. (2016, 2017) demonstrated that when predators are administered locally to a confined area, such as in rats’ lungs rather than in the blood, the predators managed to reduce the pathogen load. The lack of effect in the blood could simply be explained by the inability of predators to locate their prey when directly injected into the vascular system. Alternatively, the authors assumed that the increase in pro-inflammatory cytokines, chemokines, and innate immune cells in the blood due to predatory bacteria and pathogens could eliminate predators before they could act effectively on the pathogen. Thus, therapy using predatory bacteria can be effective for infections occurring in « immune privilege » sites (sites in the body that are able to tolerate the introduction of antigens without provoking an inflammatory immune response), for example, urinary tract infections (Shatzkes et al., 2017). Other examples are listed in the supplementary information 4.

Abundances and distribution of the BALOs

In the analysis of the absolute abundance of the different BALO families (in copies per mL, measured by qPCR) disregarding the month and the depth, the most represented family of BALOs in the three lakes corresponded to the Peredibacteraceae, with an abundance reaching up to 1.62 x 105 gene copies per mL. In contrast, Bdellovibrionaceae and Bacteriovoracaceae were on average 10,000 times lower in abundance than the Peredibacteraceae, with maximum concentrations reaching 4 and 1.25 x 101 copies per ml, respectively. Compared to total bacteria also quantified using qPCR or FCM, the Peredibacteraceae represented up to 7.12 % of total bacteria while Bacteriovoracaceae and Bdellovibrionaceae accounted for less than 0.05 % of the bacterial community. The highest concentrations were always recorded in the free-living bacterial fraction. No evident seasonal variations were recorded here. When discriminating the three families at the two distinct depths, i.e., the surface (2 m, 2.5 m or 3 m depending on the lake) vs. deeper waters (45 m or 50 m depending on the lake), (i) Peredibacteraceae were the most abundant (with 100 to 10000 times more copies per mL than Bdellovibrionaceae and Bacteriovoracaceae), (ii) Peredibacteraceae were generally more abundant at the surface compared to depth. On the other hand, we observed an opposite trend for Bdellovibrionaceae and Bacteriovoracaceae, in particular for Lake Bourget (Fig. 1).

Relationships between BALOs, total bacteria, and other environmental data

Using the relative abundance of BALOs families and environmental data obtained from the in situ surveys of peri-alpine lakes (e.g., http://www6.inra.fr/soere-ola/), a CCA was conducted to assess the relationships between BALOs and their biotic and abiotic environment (Fig. 2). The first two axes of the CCA (CCA1 and CCA2) explained 53.1% of the total variance. Bdellovibrionaceae displayed clear links with conductivity (p<0.05), and ammonium concentration (p<0.05), whereas Peredibacteraceae displayed clear links with pH (p<0.05), dissolved oxygen (p<0.05) and temperature (p<0.05). Comparatively to the two other families, no significant relationships were found for the Bacteriovoracaceae with any of the environmental factors tested here. Moreover, the two distinct water layers, i.e., the surface (<3 m depending on the lake) vs. deeper waters (>45 m depending on the lake), could be separated (Fig. 2). The analysis suggested that Bacteriovoracaceae are more abundant in deep-waters (p<0.05) and driven by ecological factors specific to this part of the water column. By contrast, Peredibacteraceae were more abundant in near-surface waters (p<0.05) and driven by environmental factors more specific to this layer (such as dissolved O2, chlorophyll a, and higher temperature). As for Bdellovibrionaceae, the repartition seemed to be less specific to the surface vs. the deeper waters (p > 0.05).

BALOs’ probable role and diversity in peri-alpine lakes

BALOs were found in each lake and at each depth investigated, whatever the period of the year sampled. While this study did not assess the role of BALOs in the microbial loop and lake functioning, it is already known from other studies that these bacteria are likely to be important bio-agents of mortality (Williams et al., 2016). It is noteworthy however that very few studies have focused on the role and effect of such predatory bacteria on the bacterial community of natural or man-made environments, and, the understanding of bacterial mortality has been mainly and mostly based on the study of viruses and protists so far (Kandel et al., 2014). Unlike viruses and protists, BALOs predation is not dependent on the physiology or the size of the prey (Chauhan et al., 2009a). Additionally, BALOs are ubiquitous in nature (Williams et al., 2016). Thus, predation by BALOs adds a new dimension to the recycling of organic matter through the microbial loop. Both viruses and BALOs recycle nutrients via the microbial loop, however the recycling mechanisms are different. Viral lysis results in the release of the entire intracellular contents of the prey into the environment, while BALOs consume most of the prey content, hence releasing few nutrients in the environment. Saying that, BALOs yield a higher energetic value since they are filled with nutrients, therefore, when other organisms graze on them, the nutrient uptake efficiency is higher (Williams et al., 2016). Regarding their diversity, BALOs form highly heterogeneous groups with a large phylogenetic diversity (Davidov et al., 2006a). We managed to detect in peri-alpine lakes the usual BALOs already found in the current bibliography, although we used a fingerprinting approach for which many biases are associated. We are aware indeed that DGGE bands only reflect the microorganism populations found at relatively high concentration. Additionally, bands can co-migrate in the DGGE gel, thereby the numbers of bands can be over- or underestimated (Berdjeb et al., 2011c). Definitely, a high throughput sequencing approach will reveal better the hidden diversity of these BALOs. Therefore, highly specific primers for each BALOs families should be designed with a fair amount of degeneracy in order to limit non-target region binding but at the same time to maximize taxa detection (Elbrecht et al., 2018). In our study, we used non-degenerate primers for the PCR-DGGE and a cloning sequence approach, therefore we might have missed some BALOs. Another very important point that we should emphasize about is the taxonomy assignment of BALOs present in 16S databases. Since early 2000, BALOs taxonomy has changed. Baer et al. (2000) reclassified Bdellovibrio stolpii and Bdellovibrio starrii into a new genus, Bacteriovorax. Then Davidov and Jurkevitch (2004), reclassified Bacteriovorax starrii as Peredibacter starrii, hence creating a new family, the Peredibacteraceae family. At the same time Baer et al. (2004) proposed to reclassify salt-water Bdellovibrio sp. as Bacteriovorax marinus and Bacteriovorax litoralis. At last, Koval et al. (2015) redirected salt water BALOs into a new genus Halobacteriovax, creating a new family, the Halobacteriovoraceae. As a result, these adjustments have caused a few confusions in 16S rRNA databases. Typically, when working with arb-SILVA SSUParc release number 132 (Quast et al., 2013) we encountered Peredibacter and Halobacteriovorax grouped in the Bacteriovoracaceae. Furthermore, some sequences were assigned to Bdellovibrio sp. At the beginning of the discovery of BALOs, any found sequence was cataloged under the Bdellovibrionaceae family. Lately, some efforts were made to assign correctly these sequences but there is much work to be done. Today again, one cannot determine whether some sequences belong to Bdellovibrio, Bacteriovorax, Peredibacter, or Halobacteriovorax.

Peredibacteraceae are the most abundant BALOs family in peri-alpine lakes

The Bdellovibrionaceae displayed little diversity in peri-alpine lakes. This result is in agreement with the study of Li and Williams (Li and Williams, 2015) who also reported that the population structure of the Bdellovibrionaceae differed from a lake to another. The Bacteriovoracaceae seem to be more diverse in saltwater than in freshwater (Davidov and Jurkevitch, 2004). While the Peredibacteraceae may be not a much-diversified family according to our study, these bacteria were found in higher concentrations compared to both the Bdellovibrionaceae and Bacteriovoracaceae. Indeed, the Peredibacteraceae isolated from freshwater and soil and described by Pineiro et al. (2004), constituted the most abundant family for all the conditions studied (within the three lakes, depths, different fractions and different sampling periods). This first result suggests that the Peredibacteraceae are well adapted to peri-alpine lake ecosystems, either by being a generalist or a versatile hunter regarding the heterotrophic bacteria present or by preying on bigger preys, thus growing faster and making more descendants. The number of preys present in the environment and the differential use of these preys (Kandel et al., 2014) affect the abundance of one population to another. Environmental factors such as temperature and salinity can also affect the distribution and abundance of BALOs families, and here the Peredibacteraceae were more correlated to temperature than the Bdellovibrionaceae or the Bacteriovoracaceae. In addition, it is known that the presence of a variety of predators such as protists (i.e. the nanoflagellates or the ciliates), metazooplankton and bacteriophages can affect the survival and the growth of bacteria within the ecosystem. In fact, these microorganisms can play a significant role in controlling bacterial populations (Pantanella et al., 2018).

Low abundance of BALOs may not be indicative of a weak functional role

The study of the abundance of the Bdellovibrionaceae and the Bacteriovoracaceae in aquaculture systems reported concentrations between 103 and 106 cells per mL (Kandel et al., 2014). These results combined with our findings could suggest that these two families have a low impact on the community of heterotrophic bacteria in peri-alpine lakes. However, recent studies have also shown that a low abundance of BALOs is not necessarily an evidence towards a lower functional impact on prey dynamics (Richards et al., 2012; Welsh et al., 2016; Williams et al., 2016). Hence, despite the very low abundances of the Bdellovibrionaceae and the Bacteriovoracaceae we found, their functional role may not be negligible. Moreover, the number of native BALOs in the environment is reported to be low (Chauhan et al., 2009b). In fact, it has been suggested that BALOs rarely dominate continuously from a numerical point of view but form reasonably abundant populations that fluctuate over time (Kandel et al., 2014). For example, a formation of a bacterial hotspot may alter the structure and the abundance of BALOs in an ecosystem at any time. This led Williams et al. (2016) to hypothesize about the “seed bank” theory. The theory implies that when some conditions are met, BALOs could switch from a state of inactive and sparse to a state where they are highly active and abundant, to the point of becoming dominant for a limited period of time. Our previous study about ssDNA viruses and their boom and bust dynamics reinforce this idea (Zhong et al., 2015). Relatively closed ecosystems such as ponds are usually rich in organic matter, resulting in high concentrations of heterotrophic bacteria that can favor the growth of BALOs populations. For example, the number of heterotrophic bacteria in shrimp ponds is 10 to 100 times greater than in natural coastal waters. BALOs react to high prey biomass densities, thus increasing their abundance (Wen et al., 2009), and can become invasive since they have a very high adaptability to different environments (Yu et al., 2017).

Table of contents :

Chapitre I : Etat de l’art sur les BALOs et objectifs de la thèse
Bactéries prédatrices : zoom sur les Bdellovibrio et organismes apparentés
1. Introduction
2. Caractéristiques générales des BALOs
3. Aspects phylogénétiques
4. Cycle de vie
4.1. Phase d’attaque
4.2. Mode endobiontique
4.3. Mode épibiontique
5. Prédation
5.1. Hôte indépendant, BALOs saprophytique ou axénique
5.2. Prédateur à Gram positif
5.3. Les autres caractéristiques de la prédation des BALOs
6. Rôle écologique
7. Applications (biotechnologie et médecine)
8. Conclusion
9. Encadrés
Bdellovibrio and like organisms: The smallest cellular hunters of the microbial world
1. Introduction
2. What makes BALOs so special to be in the spotlight?
3. BALOs phylogeny
4. Life cycle of BALOs
Box 1: Predators, micropredators, parasitoids or parasites?
4.1. Attack phase (AP)
4.2. Transition phase
4.3. Periplasmic life cycle
4.4. Epibiotic life cycle
5. BALOs predation strategies
5.1. Predation spectrum and preference
Box 2: A preference for Gram-negative prey?
5.2. Factors acting on predation
5.3. Obligate predation and host independence
6. Ecological features of BALOs
6.1. Habitats, abundance and distribution of BALOs
Box 3: Statistical methods to identify co-occurring taxa
6.2. BALOs as “population balancer” ?
6.3. The hunter hunted
7. Biotechnological applications of BALOs
7.1. Why use BALOs to fight pathogens?
7.2. Understanding pathogen predation of BALOs in vivo
8. Conclusions
Box 4 Examples of successful applications
9. Acknowledgements
Objectifs de la thèse
Chapitre 2 : Les BALOs sont-ils présents et diversifiés dans les grands lacs péri-alpins ? 
Diversity, dynamics and distribution of Bdellovibrio and like organisms in perialpine lakes
1. Introduction
2. Results
2.1. Primer selection
2.2. Abundances and distribution of the BALOs
2.3. Relationships between BALOs, total bacteria, and other environmental data
2.4. Genetic structure
2.5. Diversity
3. Discussion
3.1. BALOs’ probable role and diversity in peri-alpine lakes
3.2. Peredibacteraceae are the most abundant BALOs family in peri-alpine lakes
3.3. Low abundance of BALOs may not be indicative of a weak functional role
3.4. BALOs and environmental factors
3.5. BALOs’ provenance and form
4. Conclusions and perspectives.
5. Materials and Methods
5.1. Study sites and sampling strategy
5.2. DNA extraction and PCR primers
5.3. FCM
5.4. DGGE
5.5. DNA purification, cloning and sequencing
5.6. Sequences processing, alignment and phylogenetic analysis
5.7. Accession number(s)
5.8. Data analysis of abundance in relation to environmental data
6. Supplementary data
Bdellovibrio and like organisms in Lake Geneva: An unseen elephant in the room? 
1. Introduction
2. Materials and methods
2.1. Study site
2.2. Sampling strategy
2.3. DNA extraction
2.4. PCR and sequencing
2.5. Bioinformatics pipeline
2.6. Statistical analysis
2.7. Phylogeny
3. Results
3.1. Diversity
3.2. Phylogeny
3.3. Distribution and dynamics
3.4. Relationships between BALOs and other bacteria
4. Discussion
5. Data Availability Statement
6. Supplementary data
Chapitre III : Quelle est la diversité réelle des BALOs et leurs abondances dans divers environnements ?
New 16S rRNA primers to uncover Bdellovibrio and like organisms diversity and abundance
1. Introduction
2. Materials and Methods
2.1. BALOs primer design workflow for Illumina sequencing technology
2.2. BALOs primer design workflow for quantitative PCR
2.3. Bdellovibrio and like organisms (positive control) strains and culture
2.4. Negative control of bacteria strain and culture
2.5. Environmental samples for PCR and qPCR tests
2.6. DNA extraction
2.7. PCR amplification (primers optimization and Nano MiSeq run preparation)
2.8. qPCR amplification, cloning-sequencing and taxonomic assignation
2.9. Bioinformatics pipeline
2.10. Phylogeny
3. Results
3.1 qPCR primers specificity check and Sanger sequencing results
3.2 PCR primers specificity check and 2×250 NanoV2 MiSeq results
4. Discussion
5. Conclusion
6. Data accessibility
7. Supplementary data
Constats
Exploring the diversity, abundance and structure of Bdellovibrio and like organisms in four contrasted ecosystems over a year
1. Introduction
2. Materials and Methods
2.1. Study sites, sampling and environmental descriptors
2.2. Molecular analyses
3. Results
3.1. BALOs abundance, distribution and dynamics
3.2. Relationships between BALOs and environmental variables
3.3. OTUs diversity and structure
4. Discussion
5. Conclusion
6. Data availability
7. Supplementary data
7.1. DNA extraction
7.2. qPCR standard curves
7.3. PCR and Next-generation sequencing
7.4. Bioinformatic pipeline
7.5. Statistics
7.6. Phylogeny
Chapitre IV : Un écosystème modèle, le Léman ?
Bdellovibrio sp: An important bacterial predator in Lake Geneva?
1. Introduction
2. Material and Method
2.1. Prey isolation, selection, and culturing from different origins
2.2. Sampling and enrichment of BALOs
2.3. Predator isolation and growth on double-layered agar plates
2.4. DNA extraction
2.5. PCR amplification
2.6. Cloning, Sanger sequencing and taxonomic assignment
2.7. Phylogeny
2.8. Data accession numbers
2.9. Predatory spectrum experiment
3. Results
3.1. Characterization of the prey and predators
3.2. Predation spectrum (host-range experiment)
4. Discussion
5. Conclusion
6. Supplementary data
Short-term dynamics of Bdellovibrio and like organisms in Lake Geneva in response to a simulated climatic extreme event
1. Introduction
2. Materials and Methods
2.1 Experimental set up
2.2. Sampling strategy and analyzed parameters
2.3. DNA extraction
2.4. Quantification of BALOs by qPCR
2.5. Flow cytometry analysis
2.6. Statistics
3. Results
3.1. BALOs abundance and dynamics
3.2. Relationships with abiotic factors
3.3. Relationships between biotic variables
4. Discussion
5. Conclusions
6. Acknowledgements
7. Supplementary data
Chapitre V : Discussion et perspectives
La présence des BALOs
Références

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