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Streptomyces scabies is the causative agent of the common scab (CS) disease on tuber and root crops (potatoes, radishes, beets,…). Studies on S. scabies 87-22 are focused on understanding the molecular mechanisms and the molecules that confer this strain its virulent properties. The accessibility of its genome sequence and its subsequent analysis helped further revealing new strain-specific virulence determinants and additional features for this phytopathogen. 

The first objective of my Master II thesis is in line with previous works that is, deepening the knowledge related to the mechanisms involved in the molecular interactions existing with its host plants. This was performed by accurate genome mining of S. scabies. The biosynthetic gene clusters (BGCs) coding for (secondary) specialized metabolites predicted via the antiSMASH software have been subjected to a comparative study in order to assess, in terms of quantity and diversity, the cryptic BGCs for which no homologous BGC is known so far and therefore involved in the production of unknown compounds. Our work revealed that S. scabies possess 45 BGCs; beyond the 17 known BGCs mainly including siderophores and phytotoxins next to the core metabolites, we revealed 28 uncharacterized cryptic BGCs with poor or no homology to the currently known BGCs. Some of these BGCs might be involved in the production of cryptic new virulence key determinants, which will be confirmed or infirmed by further investigations.
The second objective of my thesis was to analyze the expression of the genes composing these 45 BGCs under culture conditions triggering the production of the main virulence determinant thaxtomin A. This work was performed by a transcriptomic study on RNA extracted when S. scabies was cultivated on minimal media containing the best known virulence triggering factors, namely cellobiose and cellotriose. With this approach we noticed that most plant-associated compounds of S. scabies followed the same transcriptional response as thaxtomin phytotoxins. The expression of most siderophore BGCs was also significantly enhanced thereby highlighting the crucial role of iron acquisition in virulence. Interestingly, the expression of several cryptic BGCs, and therefore, new molecules, was also drastically modified under virulence conditions suggesting that other and yet unknown molecules would play a significant role in the plant colonization/interaction by S. scabies.
Finally, the third objective of this work is related to the classification of S. scabies as a plant pathogen. Indeed, although CS is responsible for significant economic losses, the symptoms mostly affect the visual appearance of the vegetable, and the disease has never been reported as lethal. Could S. scabies instead contribute to the protection of its host against much more harmful pathogens by secreting antimicrobial compounds? In this work we therefore aim to provide the first evidence for answering the challenging question: “Is Streptomyces scabies really a plant-pathogen or instead a plant-protecting bacterium?”.

In order to answer this question, we assess the potential of S. scabies to produce bioactive compounds with antifungal and anti-oomycete activity by i) genome mining (also performed for the transcriptomic study), and ii) bioactivity assays. Our main focus was on the production of bioactive compounds against Phytophthora infestans - the oomycete responsible for late blight that also affects potato crops. In addition, interaction of S. scabies in antagonizing tests with ascomycetes plant pathogens such as Alternaria solani, Gibberella zeae, and Fusarium culmorum was studied as well. We reveal that S. scabies is able to produce antimicrobials (both diffusible and volatile compounds) against all pathogens tested. The identification of these compounds is key to fully understand the role of S. scabies as host for root and tuber plants.

Sciences du vivant > Microbiologie

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The studies of the bacterial communities are increasingly popular. Thanks to the continuous decrease in price of NGS services, curiosity is the limit. It is reflected in the diversity of the metabarcoding data available. Recently a collaborative Earth Microbiome Project had begun a creation of Earth’s multiscale microbial diversity catalogue unifying the effort of almost 100 independent studies for standardization of the protocol for bacterial communities analyses. However, in the public databases there is a substantial amount of the metabarcoding data that were generated throughout the years with the use of different sequencing primers targeting different hypervariable regions. 
The information about bacterial communities compositions accumulated in those metabarcoding samples could serve e.g. for identification of the origin of the sample. This work aims at establishing a base process for combining the analysis of the metabarcoding data obtained using various protocols. In the process of selection, out of over a million sequencing runs, 1567 individually processed paired-end reads samples were merged into 45 fine-scaled categories falling into four general datasets: animal-, animal-gut-, environment-, and plant-related. Next, they were processed using popular QIIME2 software without OTU clustering. Three general databases containing 16S rRNA taxonomic information, and their efficacy at five taxonomic ranks, have been tested in order to optimize the taxonomic identification of amplicon sequence variants. The above-mentioned datasets were tested for classification accuracy using two different dimensionality reduction techniques, Principal Component Analysis and Linear Discriminant Analysis applied on the similarity/dissimilarity matrices obtained separetly from an abundance and presence/absence matrices. The aptitude of machine learning in establishing the taxonomic-based classification of the sample sources has been tested with four different algorithms, radial SVM, Naive Bayes, Random Forest and k-Nearest Neighbours. The LDA transformed similarity matrix created at Order rank provided the best and most confident classification with corrected accuracy of 97.6%. Additionally, to examine whether there exist taxonomic relationships among the microorganisms detected in the aforementioned studies, the association rule learning algorithms ‘Apriori’ has been utilized. Number of co-occurrences of microorganisms on different taxonomic ranks was detected and several different taxa forming highly connected nodes were observed. Those taxa can be regarded as putative keystone taxa and considered for further investigation in different niches.

Sciences du vivant > Microbiologie

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The members of the Streptomyces genus are mainly known for their high potential to produce secondary metabolites such as antibiotics, anti-cancer drugs, volatile organic compounds, herbicides. Mostly saprophytic, few exceptions have a phytopathogenic lifestyle, with S. scabies as a model species. Research on the pathogenicity of this bacteria - the predominant causal agent of the common scab (CS) disease – has begun more than 20 years ago. The phytotoxin thaxtomin A is the main virulence factor directly related to its pathogenic lifestyle, however many molecular mechanisms still remain misunderstood and ignored. Based on computational regulon prediction and proteomics, novel gene/protein candidates involved in the development of the virulent behaviour of S. scabies have been identified. In particular, a new hypothetical signalling pathway suggesting a tight link between the cello-oligosaccharides transport and the access to the host reservoir of starch has been proposed.
The principal aim of this Master thesis was to investigate the role of two actors supposed to be involved in this pathway - the MalR regulator and the cytoplasmic component of the AfsQ1-AfsQ2 TCS -, evaluating the effects of their deletion through the PCR-targeting strategy. During this Master thesis we showed that these two important regulators certainly participate in the pathogenic lifestyle of S. scabies. The absence of MalR prevents S. scabies from perceiving the cellobiose as a signal to trigger virulence, while afsQ1 deletion resulted in increased toxin production rates, in a significant impairment of A. thaliana growth and in more severe pitting and necrosis of potato tuber slices.
A further objective of this work aimed to generate new strains of S. scabies with improved thaxtomin production yields. Indeed, the properties of this phytotoxin made it a prime candidate as bioherbicide, however, nowadays the production costs are still too high for a large-scale application. Earlier works have shown that the mutation of either cebR or bglC genes resulted in an overproduction of thaxtomin A, therefore we have investigated the possibility that combining the deletion of both genes would turn in even higher production rates. The new generated strains are capable of producing significant toxin rates, however, although significant, did not exceed those of the single mutants.

common scab disease --- plan pathogen --- regulatory networks --- plant-microbe interactions --- herbicide --- Sciences du vivant > Microbiologie

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The cane toad (Rhinella marina) is a performant invasive species that has spread throughout sub-tropical and tropical Australia. Introduced in 1935 in a failed attempt to control agricultural pests, cane toads have since posed a threat to native ecosystems. By a chemical self-defence mechanism, they are poisonous to most Australian anurophagous at all life stages. The cane toad toxins are a complex mixture dominated by bufadienolides, a class of cardiotoxic steroids. The cane toad chemical ecology has become an important part of the fight against this invasive species because it has brought key knowledge that inspired practical control methods. Bufadienolides are principally stored in the form of stable bufotoxins in the adult cane toad parotoid glands. The secretion of parotoid toxins provokes the ex situ enzymatic hydrolysis of bufotoxins into more potent bufadienolides called bufagenins. In laboratory conditions, parotoid gland-associated bacteria were found to mediate either the degradation or transformation of bufagenins into oxidised or hydroxylated analogues respectively. However, road-killed cane toads were found to remain toxic to native predators in field studies. 
This research project intended to shed some light on the natural evolution of bufadienolides and evaluate the importance of microbiological degradation and transformation within the natural substrate. Firstly, investigations were conducted into the chemistry of parotoid glands of four road-killed cane toads in decomposition. They revealed that bufadienolide profiles first dominated by bufotoxins quickly evolved to the profit of bufagenins in a similar way than for parotoid secretion. The subsequent profiles remained unchanged and no evolution was observed. Only one hydroxylated analogue (11-hydroxymarinobufagenin) was detected but at a constantly low level. Then, parotoid gland-associated bacteria that were previously proved to degrade or transform bufagenins were challenged with marinobufagenin. The results helped describe and differentiate the different microbiological processes: the biodegradation of bufagenins is an oxidation mediated by gram negative bacteria that follows a sequence to produce oxidised scaffolds; the biotransformation of bufagenins is a hydroxylation mediated by gram positive bacteria independently yielding hydroxylated analogues. Finally, strains were isolated from the four road-killed cane toad parotoid glands and challenged with marinobufagenin. Each bufadienolide profile was compared with reference results as to determine if it was originated from microbial degradation or transformation. The comparison confirmed that parotoid glands are a source of degrading and transforming bacteria. Consequently, this research project demonstrated that bufagenins were the agent of the enduring toxicity of road-killed cane toads and remained stable despite the presence of biodegrading and biotransforming bacteria within the parotoid glands. This area of study has still got the potential to bring key knowledge in the general understanding of bufadienolide metamorphosis and inspire new ways to control the Australian cane toad.

Rhinella marina, cane toad, road-killed, bufadienolides, biotransformation, biodegradation --- Sciences du vivant > Microbiologie --- Sciences du vivant > Biochimie, biophysique & biologie moléculaire

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The cyclic lipopeptide (CLP) surfactin is secreted by Bacillus spp. such as Bacillus velezensis, a plant growth promoting rhizobacteria (PGPR). This molecule has biological functions among which the ability to stimulate the induced systemic resistance (ISR) in several plant models. This phenomenon could be part of the transition to sustainable agriculture since it can help the plant to face many pathogens infections. However, the molecular basis of ISR induction by surfactin is still poorly understood. In this context, a structure-activity approach of surfactin has been adopted to get deeper insights into the relation between this CLP and ISR . Thus, the aim of this thesis is to evaluate the impact
of structural changes in the plant immunization potential of surfactin. For this purpose, naturally occurring variants of surfactin, pumilacidin and lichenysin, have been chosen. These CLPs vary in fatty acid chain length and in peptide composition. In order to deepen our knowledge in the induction of ISR in planta, the reactive oxygen species (ROS) burst and the stimulation of the transcriptional factor MYB72, two plant immunity markers, have been studied. To achieve these objectives, surfactin, pumilacidin and lichenysin have been produced and purified in order to test their ability to trigger ROS burst and myb72 stimulation in addition to their activity as ISR elicitors in Arabidopsis thaliana L. (Arabidopsis), the plant model used in this study. In this work, we firstly demonstrate that the structure of surfactin is determinant for ISR induction in Arabidopsis. Then, regarding ISR markers, our results suggest that myb72 could be an interesting ISR marker where ROS burst alone is not sufficient to trigger ISR. Moreover, we prove that the change of one single amino acid in the peptide ring of the CLP can alter the activity of surfactin. Furthermore, this work provides new insight into ISR induction and in particular on the role of the enzyme, β-glucosidase
BGLU42 in relation to myb72.

Surfactin --- Pumilacidin --- ISR --- Lichenysin --- Arabidopsis --- Bacillus --- Structure --- Sciences du vivant > Biochimie, biophysique & biologie moléculaire --- Sciences du vivant > Microbiologie