Bahlawane Discussion

The discussion is formulated, as the entire thesis, along with the two major investigations done during this study. These are the study of the regulatory mechanism of wg genes expression in the first part and of Rem expression in the second part. An emphasis is given to the role of MucR in both regulation paths. Finally, the conclusion will try to point out the consequences of an inter-connected regulation and be closed on the role of MucR for the symbiosis efficiency in S. meliloti. I. Regulation scheme of galactoglucan synthesis in low and high Pi Published, also already edited II.Regulation scheme of motility genes in S. meliloti. Motility and chemotaxis offers micro – organisms a distinct advantage when faced with starvation, toxic substances or a requirement to approach the appropriate host. However, the motility machinery places high energy demands on the cell. Bacteria develop a strict regulation network to control the expression of motility – related genes to av oid inappropriate energy expenditure. Bacteria also invoke alternative strategies for the regulation of motility when faced with starvation or reduced nutrient conditions. In some bacteria, e. g., Serratia liquefaciens and Escherichia coli, this strict regulation results an increase of ability to migrate when faced with reduced nutrient conditions (Sharma, Anand, 2002). Apparently the strategy is to engage the motility machinery only in response to stress / starvation conditions by moving towards a better environment. In other bacteria, e. g. S. meliloti, the strict regulation results migration toward chemo-attractants under nutrient sufficient or excess conditions and the development of the vegetative state and biofilm upon encountering stress/starvation conditions (Wei, Bauer, 1998).Thus, S. meliloti was found to be motile throughout active growth, when nutrients are readily available, and to become non-motile upon transition to the stationary phase (Rotter et al. , 2006). The clu e of this regulation remained unknown up to the present. Some advances have been performed by the discovery of Rem (Rotter et al. , 2006), a Class Ib regulator, which expression follows the cell motility. Recently, Hoang et al. (Hoang et al. , 2008) demonstrated that quorum sensing (QS) regulated motility in strain Rm8530 through the regulation of visNR expression by ExpR by using an unknown mechanism.1. Modulation of rem expression Since rem expression was found to be growth – dependent and that visN expression was constant all through the growth, new regulators of motility were expected (Rotter et al. , 2006). In the present study, we demonstrated that MucR affects motility by regulation of rem expression. Binding of MucR to the promoter region upstream of rem was shown and a sequence sharing similarities with the MucR binding site consensus (Bertram-Drogatz et al. , 1998) was identified in this region.However, further investigations suggested that sequences, downstream of this consensus, could be required for MucR binding to the rem upstream region. Since MucR inhibition was increased by a mutation in rem, we speculated that the ratio of Rem / MucR levels in the cell may contribute to the regulation of motility in S. meliloti. In RU11/001, Rem was found to be present in great amount in cells at low density (Rotter et al. , 2006) and slowly disappear during the exponential growth. We did not retrieve a much higher rem transcription level at low cell density in our wild type Rm2011.However, we cannot exclude that post – transcriptional regulation is responsible for the accumulation of Rem protein in Rm2011 at low cell density. If high levels of Rem are present in the cell, the effect of MucR on regulation of rem appears to be negligible. Consistent with this postulation, we observed that at low cell densities, the expression of rem promoter was inhibited in the presence of an intact mucR locus and a disrupted rem locus, but not in the presence o f a functional rem locus.In such scenario, the quantity of Rem within the cell in a culture of low cell density might be high enough to completely mask the effect of MucR, while at higher cell density, the higher quantity of MucR within the cell might be sufficient to inhibit transcription of rem. Another explanation for the difference of rem expression pattern between the strain RU11/001 and Rm2011 might be the expR status of these two independent wild types. Indeed, Rm2011 exhibits an insertion element within expR (Pellock et al. , 2002), leading to a non – functional gene and consequently a deficient quorum- sensing (QS) system.It was previously shown that the Sin / ExpR quorum sensing system (Marketon et al. , 2002;Marketon et al. , 2003) down-regulates motility and chemotaxis genes and that the visN-visR operon was a target for the LuxR-type regulator ExpR (HOANG 2004, HOANG 2008). We could confirm the inhibition of visN expression by AHL-activated ExpR and its relay to the rem expression. Moreover, McIntosh demonstrated binding of ExpR to the promoter region of visN in the presence of AHLs implying that ExpR directly regulates transcription of visN-visR (personal communication).While rem expression levels are regulated by several regulators, including ExpR, VisN and VisR, as demonstrated in this study and elsewhere (Hoang et al. , 2008;Rotter et al. , 2006) it is unknown what factors affect expression of the autoregulated mucR gene (Muller et al. , 1993). However, MucR appears to function as a repressor, both in the presence and absence of a functional Sin quorum sensing system (Bahlawane C. et al. , 2008;McIntosh et al. , 2008), suggesting that the regulation of mucR expression is not dependent on quorum sensing, although this remains to be confirmed.2. Requirement for swimming and swarming motility This study highlights another interesting aspect of motility in S. meliloti, namely, swarming, but not swimming, depends on the presence of a functio nal quorum sensing system and galactoglucan production. The requirement of exopolysaccharides for swarming was recently highlighted in a large scale analysis of Tn5 mutants exhibiting swarming defects in Rhizobium etli (Braeken et al. , 2008). The requirement of a functional quorum sensing for swarming was likewise found in Rhizobium etli (Braeken et al. , 2008;Daniels et al., 2006). However, this strain exhibits two different quorum sensing systems CinIR and RaiIR, with a much brighter QS signal molecules spectrum. We demonstrated that in S. meliloti, only those strains with an intact ExpR / Sin quorum sensing system and capable of galactoglucan and flagella production showed the ability to swarm. Even if MucR modulated the expression of the regulator of motility, the swimming ability of a mucR, expR – mutant (Rm101) was not affected. Therefore, swimming required flagella production, but not the ExpR/Sin system and galactoglucan biosynthesis.Swimming is an individual endeavo ur, whereas swarming is the coordinated movement of a population of bacteria in high viscosity media or over a surface and has been shown to be widespread among flagellated bacteria, particularly in association with quorum sensing (Harshey, 2003;Sharma, Anand, 2002). Hence, the involvement of quorum sensing in swarming, but not in swimming, may be an important feature in coordination of motility. 3. Interconnection between both regulation networks and comparison with other strainsCoupling of the regulation of bacterial exopolysaccharide production and the regulation of cell motility has also been found in Ralstonia solanacearum (5), Vibrio cholerae (1, 34), and Salmonella enterica (7), and presumably increases the ability of the cells to interact with their prospective host cells. We found that ExpR functions as inhibitor of the master regulator of motility VisNR, in addition to its role as an activator of the expression of the galactoglucan genes in the presence of Sin AHLs and pos itively affect expression of exoK and exsH leading to the depolymerisation of succinoglycan (McIntosh et al., 2008). Moreover, MucR is shown to inhibit the motility through rem expression, in addition to its role in the activation the HMW succinoglycan production and inhibition of the galactoglucan production. This fits well to the opposite regulation for succinoglycan biosynthesis and motility found previously for S. meliloti strains (Hoang et al. , 2008;Wells et al. , 2007;Yao et al. , 2004). A model is therefore proposed (Fig. 4. 2) where the inverse regulation of succinoglycan and flagella synthesis, as well as the role of MucR and ExpR are highlighted. Figure 4.1 Regulation schem of the motility in S. meliloti Dashed lines indicate an unknown regulation path and lines indicate a direct regulation through demonstrated DNA / protein interactions. The direct effect of ExoR /ExoS has to be further analyses, expecially the interaction with visN promoter has to be elucidated. Chapter 2 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 s ystem, 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 Puh ler, 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 givin g 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 t o 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 bios ynthesis 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 clas s 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.