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dc.contributor.author
Ledermann, Raphael
dc.contributor.supervisor
Fischer, Hans-Martin
dc.contributor.supervisor
Vorholt, Julia A.
dc.contributor.supervisor
Christen, Beat
dc.contributor.supervisor
Perret Xavier
dc.date.accessioned
2017-10-30T15:12:36Z
dc.date.available
2017-10-30T14:03:23Z
dc.date.available
2017-10-30T15:12:36Z
dc.date.issued
2017
dc.identifier.uri
http://hdl.handle.net/20.500.11850/202317
dc.identifier.doi
10.3929/ethz-b-000202317
dc.description.abstract
To cope with changing environments, bacteria need to monitor thoroughly a plethora of different parameters. This includes external factors such as osmotic pressure, pH, temperature, irradiation, or nutrient availability but also internal factors such as energy state. Depending on the species, each parameter has a window, in which optimal growth occurs. Non-optimal conditions are generally referred to as stress and may result in either limited growth, dormancy, or dead of the bacterium. Although they can be very diverse, most stresses cause damage to macromolecules such as proteins, lipids (membranes), or nucleic acids. To limit the adverse effects of stress conditions, an unfavorable condition is sensed and an appropriate response is mounted. For many stressful conditions, a specific response occurs. Examples include (i) responses to reactive oxygen and nitrogen species, which result in the production of detoxifying enzymes (e.g. catalases, peroxidases, superoxide dismutases), (ii) the SOS response, mounted as reaction to DNA damage (e.g. after irradiation), which results in production of DNA repair enzymes (e.g. recombinases, excision repair endonucleases, ligases, DNA polymerases), and (iii) the heat shock response which produces e.g. chaperones to enhance protein stability and proteases to degrade misfolded proteins. Besides such specific responses, some bacteria also can mount a general stress response (GSR). GSR systems have been found in phylogenetically distinct bacteria, and likewise, the regulators of GSR systems are not related. Instead, common to GSR is cross protection. This term describes the fact that a GSR is mounted in response to a variety of stresses and results in the production of different factors and in physiological adaptations alleviating negative effects of a range of stresses. Hence, induction of the GSR by one stress condition renders the bacterium more tolerant against completely unrelated stresses as well. In α-proteobacteria, the GSR core regulators are the alternative σ factor σEcfG, its cognate anti-σ factor NepR, and the anti-σ factor antagonist PhyR. A variety of sensors, often sensory histidine kinases, are responsible for detection of different types of stresses, which ultimately lead to (direct or indirect) PhyR phosphorylation. This causes a conformational switch of PhyR, exposing its σ factor-like domain, which has a higher affinity for NepR than the actual σ factor σEcfG. Hence, phosphorylated PhyR titrates NepR away from σEcfG, which was previously bound and thus rendered inactive by NepR. Thereby freed σEcfG redirects transcription to stress response genes. A subclass of α-proteobacteria, generally referred to as rhizobia, are capable of establishing an endosymbiosis with legume plants. Thereby, bacteria enter plant roots after specific chemical cross talk and end up in plant cells of genuine plant organs called root nodules. There, they fix atmospheric dinitrogen to ammonia, which is exchanged with the plant for reduced carbon sources and other nutrients. When either ecfG or phyR is deleted, the soybean (Glycine max) symbiont Bradyrhizobium diazoefficiens fails to establish a wild type-like symbiosis. To study the role of the GSR in the B. diazoefficiens–soybean symbiosis, we first developed a set of novel genetic tools for this bacterium. These include (i) fluorescent and enzymatic tags, stably integrated into the chromosome for in planta observations of the bacteria, (ii) a streamlined system for targeted gene deletion and (iii) constructs allowing controlled inducible gene expression. With the help of these novel tools, we show that the GSR of B. diazoefficiens is mounted against a variety of stresses (salts, hyperosmosis, elevated temperature, alkaline pH). Furthermore, the GSR is also induced during early time point of symbiosis, when bacteria are trapped in curled root hairs and form microcolonies before they enter the root via the so-called infection threads (ITs). However, the GSR is neither active in rhizosphere-colonizing bacteria nor in nitrogen-fixing endosymbionts (bacteroids). Likewise, we found that the aberrant symbiosis of GSR mutants is due to their ineffective IT formation ability. The genome of B. diazoefficiens encodes a set of 11 sensory histidine kinases (HhkA through HhkK) which all comprise a characteristic HRxxN amino acid sequence motif and are candidates for activation of the GSR in stressed free-living cells and root hair-entrapped bacteria. Because single deletion mutants lacking individual hhk genes showed a wild type-like phenotype when inoculated on soybean plants, we suspected functional redundancy and started to generate multiple deletion mutants. When the symbiotic phenotype of septuple and decuple mutants was determined, we found an increasing delay in nodulation with increasing number of hhk kinase gene deletions. Furthermore, some of the multiple mutants lost the ability to mount the GSR in response to selected stresses encountered in free-living bacteria. This further supports a partial redundancy in function between different Hhk kinases in sensing the same stress. Finally, we investigated the σEcfG regulon to find gene (products) needed for proper IT formation. We deleted a series of candidate genes and found that deletion or misregulation of two σEcfG-controlled genes involved in trehalose biosynthesis, otsA and otsB, phenocopied ΔphyR and ΔecfG mutants. OtsA is a trehalose-6-phosphate synthase using glucose-6-phosphate and UDP-glucose as substrates while OtsB is a trehalose-6-phosphate phosphatase. Besides otsA and otsB, B. diazoefficiens encodes two alternative trehalose biosynthesis pathways. However, mutants with all respective genes deleted were not disturbed with respect to trehalose accumulation in free-living cells and symbiotic performance. On the other hand, overexpression of Escherichia coli treF, coding for a cytoplasmic trehalase, in a B. diazoefficiens wild-type background resulted in a similar phenotype as deletion of otsA and/or otsB. Furthermore, complementation assays with otsA and otsB controlled by different promoters indicated that fine-tuned spatio-temporal expression of these genes mediated by σEcfG-dependent regulation is crucial for functional symbiosis. Overall, this thesis documents that the B. diazoefficiens ΔecfG mutant phenotype in symbiosis with soybean is due to the lack of otsA and otsB expression. This pathway represents the major source of trehalose in B. diazoefficiens. Trehalose is needed during IT formation but its synthesis is detrimental for nitrogen fixation in bacteroids. Likewise, the natural σEcfG-dependent regulation of otsA and otsB ensures just this correct spatio-temporal expression.
en_US
dc.language.iso
en
en_US
dc.publisher
ETH Zurich
en_US
dc.title
Role of General Stress Response in Trehalose Biosynthesis for Functional Rhizobia-Legume Symbiosis
en_US
dc.type
Doctoral Thesis
ethz.size
220 p.
en_US
ethz.grant
The role of Bradyrhizobium japonicum extracytoplasmic function (ECF) sigma factor EcfG in formation of an effective symbiosis
en_US
ethz.identifier.diss
24611
en_US
ethz.publication.place
Zurich
en_US
ethz.publication.status
published
en_US
ethz.leitzahl
ETH Zürich::00002 - ETH Zürich::00012 - Lehre und Forschung::00007 - Departemente::02030 - Dep. Biologie / Dep. of Biology::02520 - Institut für Mikrobiologie / Institute of Microbiology::08818 - Fischer, Hans-Martin (Tit.-Prof.)
en_US
ethz.grant.agreementno
153446
ethz.grant.fundername
SNF
ethz.grant.funderDoi
10.13039/501100001711
ethz.grant.program
Projektförderung in Biologie und Medizin (Abteilung III)
ethz.date.deposited
2017-10-30T14:03:24Z
ethz.source
FORM
ethz.eth
yes
en_US
ethz.availability
Embargoed
en_US
ethz.date.embargoend
2020-10-30
ethz.rosetta.installDate
2017-10-30T15:12:43Z
ethz.rosetta.lastUpdated
2018-11-05T23:52:03Z
ethz.rosetta.versionExported
true
ethz.COinS
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