Sinorhizobium meliloti is a nitrogen-fixing endosymbiont of the legume Medicago sativa commonly known as alfalfa. It uses flagellar rotation and chemotaxis to seek roots of host plants to inhabit. This symbiosis serves as a great model system for studying biological nitrogen fixation and plant-microbe interactions. Since alfalfa brings enormous economic value to the USA, investments into the knowledge of the chemotaxis process that initiates symbiosis have the ability to mitigate deterioration of the environment and significantly increase food supply. The chemotaxis system in the enteric bacteria Escherichia coli is well studied and has been a great resource to understanding the process in other bacterial systems including our model organism S. meliloti.
This dissertation compares and contrasts the chemotaxis features in E. coli and S. meliloti and investigates their molecular functions. Based on the understanding gained so far, we attempt to offer plausible explanations for the underlying mechanisms of the S. meliloti chemotaxis pathway. Chapter 1 describes why biological nitrogen fixation is important for agriculture and the health of our environment. This chapter also sheds light on the symbiotic relationship between alfalfa and S. meliloti, which culminates in the formation of nitrogen fixing nodules. We expound on the chemotaxis systems in E. coli and other bacteria including S. meliloti and Bacillus subtilis.
In chapter 2, we compare the distribution of C-terminal pentapeptide-bearing receptors and the adaptation proteins that they tether in E. coli and S. meliloti. The stoichiometry data show that the ratio of pentapeptide-bearing chemoreceptors to chemotaxis protein (Che)R and CheB molecules are approximately 500- and 160-fold higher in S. meliloti than in E. coli, respectively. Since not all chemoreceptors in chemotactic bacteria have and utilize the pentapeptide moiety, we investigated the S. meliloti system and observed a strong interaction between CheR, activated CheB and the isolated pentapeptides via in-vitro binding studies. On the contrary, unmodified CheB showed weak binding to the pentapeptide. Through in-vivo studies, we highlighted the physiological necessity of the pentapeptide for chemotaxis. S. meliloti strains with substitutions of the conserved tryptophan residue to alanine in one or all four pentapeptide-bearing Methyl-accepting Chemotaxis Proteins (MCPs) resulted in diminished or loss of chemotaxis to glycine betaine, lysine, and acetate, ligands sensed by pentapeptide-bearing McpX and pentapeptide-lacking McpU and McpV, respectively. The flexible linker connecting the pentapeptide to the MCPs together with the pentapeptide itself were shown to be functional on pentapeptide-lacking chemoreceptors and provided adaptational assistance to other chemoreceptors that lacked a functional pentapeptide. Based on these results, we concluded that S. meliloti employs a pentapeptide-dependent adaptation system with MCPs possessing a consensus pentapeptide motif (N/D)WE(E/N)F). Finally, we postulated that the higher abundance of CheR and CheB in S. meliloti compared to E. coli compensates for the lower number of pentapeptide-bearing chemoreceptors in the chemosensory array.
In chapter 3, we explored the putative phosphatase function of a novel protein, CheT, on phosphorylated S. meliloti response regulators. The kinase CheA phosphorylates both the sink response regulator, CheY1, and the flagellar motor interacting response regulator, CheY2. CheY1 competes with CheY2 for these phosphate groups, but we have discovered another layer of complexity to the story. Sequence comparison of S. meliloti CheT and the E. coli phosphatase CheZ shows little sequence homology. However, both proteins share a DXXXQ phosphatase motif. Phosphorylation assays performed using radiolabeled [γ-32P]-ATP revealed that CheT acts as a phosphatase of CheY1~P and accelerates dephosphorylation of CheY1~P by at least two-fold. Interestingly, we also discovered that CheT interacts with CheR, but this interaction did not affect the enzymatic activity of either protein under the examined conditions. Unexpectedly, a cheT deletion strain and strains carrying mutations in the phosphatase motif exhibit an increased swimming speed, a phenotype that does not conform with the model that the absence of CheT or its activity results in increased CheY2~P levels and reduced swimming speed. We concluded that a revised S. meliloti signal termination pathway should include CheT enhancing dephosphorylation of CheY1~P and sensory adaptation involving the yet unknown function of CheT on CheR.
While the adaptation system in S. meliloti is unexplored, this work provides first insights into fascinating deviations and similarities to the known paradigm. We have also delivered evidence that the S. meliloti signal termination system requires a dedicated phosphatase. The knowledge gained here takes us a step closer to enhance the S. meliloti chemotaxis pathway towards improved symbiosis with alfalfa and to reduce our dependence on environmentally deleterious synthetic fertilizers. / Doctor of Philosophy / Like all living things, bacteria inhabit a constantly changing environment, hence the need to take up and process this information. Bacterial cells have evolved sophisticated biological tools to tackle this challenge of detecting, responding and adapting to environmental signals like nutrients, toxins, temperature changes, light, metabolites, etc. Motile bacteria such as Escherichia coli, a gut resident microbe, and Sinorhizobium meliloti, a soil dwelling bacterium, direct their swimming behavior in response to chemical gradients within the milieu through a process termed chemotaxis. Generally, this vital process enables a bacterium to escape harmful chemicals and gravitate towards beneficial ones. However, S. meliloti specifically employs chemotaxis to locate the roots of its plant host (alfalfa) and to establish a symbiotic relationship through which the bacteria provide essential nitrogen for plant growth in exchange for nourishment. The biological tools employed by S. meliloti for chemotaxis include environmental sensing receptors called Methyl-accepting Chemotaxis Proteins (MCPs) and proteins inside the bacterial cell that transfer information from the sensors to long, helical rotating propeller structures, called flagella. Importantly, the efficiency of this process hinges on a timely termination of information flow and the ability to adapt to prevailing stimuli while maintaining sensitivity to increasing concentration gradients. This work investigates the function of the C-terminal five amino acid motif of MCPs known to be critical for adaptation in E. coli and the phosphatase activity of a novel protein, CheT, in signal termination of S. meliloti chemotaxis system.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/117273 |
| Date | 21 December 2023 |
| Creators | Agbekudzi, Alfred |
| Contributors | Biological Sciences, Scharf, Birgit, Vinatzer, Boris A., Tholl, Dorothea, Caswell, Clayton Christopher |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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