Bahlawane discussion Essay

Conclusion Taking in account the scheme of galactoglucan synthesis regulation (Fig. 4. 1) and the scheme of motility regulation (Fig. 4. 2) proposed in this study, we may try to follow the effect of such regulation paths in the life – styles of the bacteria. The last one is named as free – living microorganism or as symbiont in the plant root. In the first case, the bacteria are exposed to dryness and nutrients starvation. At this stage, the biofilm production is a pre-requisite for survival. Therefore, the EPS’s biosynthesis has to be switched ON. As it is quite improbable that the cell density is high enough to activate the quorum sensing system, we can speculate that MucR plays a key role in activating the succinoglycan biosynthesis. Since the soil exhibits very low level of Pi, galactoglucan is, at this point produced through the activation via phoB and WggR. Upon biofilm formation, the cell density probably increases and could activate the quorum sensing system, allowing the bacteria to produce more galactoglucan and swarm towards better conditions. In such conformation, the cells present within the centre of the swarming population are non motile and synthesized EPS; thus the cells present at the migration front are highly motile but do not synthesized EPS. It would be interesting to clarify whether a cell differentiation, as proposed by Soto (Soto et al. , 2002), takes place at this stage. Finally, if the host is present in the next neighbourhood the chemotactic compounds, as well as the nutrients found in the rood exudates, will attract the bacteria. When approaching the root, the moisture increases, along with the nutrients availability; so that swarming motility will be replaced by swimming motility that decline progressively. Indeed, the bacteria attached to the root, increasing the cell density that may lead to the inhibition of flagella production via ExpR / QS. Instead, EPS are produced, allowing recognition between the plant and the microbe as well as the invasion of the new synthesized infection thread. Once within the root, the bacteria will differentiate to bacteroid and start fixing nitrogen. We tried to highlight in this thesis the relevance of exopolysaccharides and motility for an efficient symbiosis. Great advances have been made the last years, leading to the identification of the quorum sensing interaction with motility. We participate in inclusion of MucR, as new regulator of motility and ExpR as requisite for swarming. However, the full understanding of the influence of motility in symbiosis establishment will require finding out which signals are inducing mucR and exoR/ exoS. Moreover, some tests have to be implemented to investigate the symbiosis establishment in more realistic conditions. Indeed, the bacteria are usually directly inoculated to the root, so that motility, via swarming or swimming is not required. Acknowledgments First and foremost, I would like to thank Prof. Dr. Alfred Puhler, Chair of the Genetics department, for allowing me use the very good infrastructure that promotes a very pleasant and conducive atmosphere during my research using performant techniques. I am especially grateful to Prof. Dr. Anke Becker, my supervisor, for giving me the chance to come back to research. Without her advices, ideas and resources, this work would not be possible and achieved. Thus, I thank her too for the freedom she gave me, as well as her support to test new ideas and her great help by conceiving and writing the manuscripts that become the pillars of this manuscript. Within the laboratory members, I would like to thank first Dr. Birgit Baumgarth who introduced me to the lab and to the investigated organism. Then, special thanks to Dr.Matthew McIntosh for the quorum sensing – related work and his help for preparing the derived publication. Furthermore, I would like to deeply thank Dr. Natasha Pobigaylo for her friendship, her helpful discussions and for giving me courage when I am about to lose it. I thank Manuela Mayer, too, for the assistance in microarray hybridizations as well as Dr. Lisa Krol, Javier Serrania and Thomas Montfort for the everyday help in the lab. Finally, I would like to thank all Exopol group members for the support and advices. Least, I would like to thank my family for their unending and heartwarming support in many ways. Special thanks to Rachida Bendaou, my mother-in-law, for her support in caring my children during my research. I would like to thank my children, Ines, Soraya and Jasmine, for filling up my life with love and happiness. I would like to apologize for the bad mood and stress situations that are unfortunately connected with such a thesis. My heartfelt gratitude to my understanding and loving husband, Naoufal, for his moral and financial support, for believing in me and for sharing the passion for science with me. Resume In order to enter symbiosis with its legume partner, Sinorhizobium meliloti has to face continual changing conditions. It has more ability to adapt quickly to the situation than the ability to face it efficiently that makes the difference in term of symbiosis efficiency. For the first interactions with its host, motility is required by S. meliloti to move towards the chemotactic compounds released by its host when exopolysaccharides (EPSs) are required later on, for the attachment to the root as well as for the invasion of the infection thread, leading to the formation of the root nodule. We focused in this study the regulatory networks leading to the coordination of motility and EPS’s production in the strain Rm2011. Depending on the phosphate concentration encountered in the environment Rm2011 synthesizes two different exopolysaccharides (EPS). Galactoglucan (EPS II) is produced under phosphate starvation but also in the presence of extra copies of the transcriptional regulator WggR (ExpG) or as a consequence of a mutation in mucR. The galactoglucan biosynthesis gene cluster contains the operons wga (expA), wge (expE), wgd (expD), and wggR (expG). Two promoters, differentially controlled by WggR, PhoB, and MucR, were identified upstream of each of these operons. The proximal promoters of the wga, wge, and wgd transcription units were constitutively active when separated from the upstream regulatory sequences. Promoter activity studies and the positions of predicted PhoB and WggR binding sites suggested that the proximal promoters are cooperatively induced by PhoB and WggR. MucR was shown to strongly inhibit the distal promoters and bound to the DNA in the vicinity of the distal transcription start sites. An additional inhibitory effect on the distal promoter of the structural galactoglucan biosynthesis genes was identified as a new feature of WggR in a mucR mutant. Motility is organized in S. meliloti in a hierarchical cascade, with Class Ia genes, encoding the major regulator of motility VisNR; controlling the expression of the class Ib gene, rem, which encodes a central regulator, activating the expression of the downstream Class II and class III genes. We could demonstrate that MucR binds a DNA sequence upstream of rem, following a different mechanism as previously observed upon binding upstream of the wg genes. By this way, MucR inhibits rem expression as well as the expression of the Rem-regulated genes such as flaF and flgG. Furthermore, we addressed a balance of the swimming and swarming abilities of several S. meliloti strains derivatives of Rm2011. We could show that all strains, able to build flagella, were swimming on low viscosity agar plates. However, swarming over high viscosity agar plates required all a functional expR / sin locus, the ability to build flagellum and the production of exopolysaccharides. Finally, we propose a model for the coordination of motility and EPSs synthesis in S. meliloti.