An Integrated Systems Approach Unveils New Aspects of Microoxia-Mediated Regulation in Bradyrhizobium diazoefficiens

The adaptation of rhizobia from the free-living state in soil to the endosymbiotic state comprises several physiological changes in order to cope with the extremely low oxygen availability (microoxia) within nodules. To uncover cellular functions required for bacterial adaptation to microoxia directly at the protein level, we applied a systems biology approach on the key rhizobial model and soybean endosymbiont Bradyrhizobium diazoefficiens USDA 110 (formerly B. japonicum USDA 110). As a first step, the complete genome of B. diazoefficiens 110spc4, the model strain used in most prior functional genomics studies, was sequenced revealing a deletion of a ~202 kb fragment harboring 223 genes and several additional differences, compared to strain USDA 110. Importantly, the deletion strain showed no significantly different phenotype during symbiosis with several host plants, reinforcing the value of previous OMICS studies. We next performed shotgun proteomics and detected 2,900 and 2,826 proteins in oxically and microoxically grown cells, respectively, largely expanding our knowledge about the inventory of rhizobial proteins expressed in microoxia. A set of 62 proteins was significantly induced under microoxic conditions, including the two nitrogenase subunits NifDK, the nitrogenase reductase NifH, and several subunits of the high-affinity terminal cbb3 oxidase (FixNOQP) required for bacterial respiration inside nodules. Integration with the previously defined microoxia-induced transcriptome uncovered a set of 639 genes or proteins uniquely expressed in microoxia. Finally, besides providing proteogenomic evidence for novelties, we also identified proteins with a regulation similar to that of FixK2: transcript levels of these protein-coding genes were significantly induced, while the corresponding protein abundance remained unchanged or was down-regulated. This suggested that, apart from fixK2, additional B. diazoefficiens genes might be under microoxia-specific post-transcriptional control. This hypothesis was indeed confirmed for several targets (HemA, HemB, and ClpA) by immunoblot analysis

Role of General Stress Response in Trehalose Biosynthesis for Functional Rhizobia-Legume Symbiosis

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

A Functional General Stress Response of Bradyrhizobium diazoefficiens Is Required for Early Stages of Host Plant Infection

Phylogenetically diverse bacteria respond to various stress conditions by mounting a general stress response (GSR) resulting in the induction of protection or damage repair functions. In α-proteobacteria, the GSR is induced by a regulatory cascade consisting of the extracytoplasmic function (ECF) σ factor σEcfG, its anti-σ factor NepR, and the anti-anti-σ factor PhyR. We have reported previously that σEcfG and PhyR of Bradyrhizobium diazoefficiens (formerly named Bradyrhizobium japonicum), the nitrogen-fixing root nodule symbiont of soybean and related legumes, are required for efficient symbiosis; however, the precise role of the GSR remained undefined. Here, we analyze the symbiotic defects of a B. diazoefficiens mutant lacking σEcfG by comparing distinct infection stages of enzymatically or fluorescently tagged wild-type and mutant bacteria. Although root colonization and root hair curling were indistinguishable, the mutant was not competitive, and showed delayed development of emerging nodules and only a few infection threads. Consequently, many of the mutant-induced nodules were aborted, empty, or partially colonized. Congruent with these results, we found that σEcfG was active in bacteria present in root-hair-entrapped microcolonies and infection threads but not in root-associated bacteria and nitrogen-fixing bacteroids. We conclude that GSR-controlled functions are crucial for synchronization of infection thread formation, colonization, and nodule development. </jats:p

Aspartate aminotransferase of Rhizobium leguminosarum has extended substrate specificity and metabolises aspartate to enable N2-fixation in pea nodules

Rhizobium leguminosarum aspartate aminotransferase (AatA) mutants show drastically reduced symbiotic nitrogen fixation in legume nodules. Whilst AatA reversibly transaminates the two major amino-donor compounds aspartate and glutamate the reason for the lack of N2 fixation in the mutant has remained unclear. During our investigations into the role of AatA we found it catalyses an additional transamination reaction between aspartate and pyruvate forming alanine. This secondary reaction runs at around 60% of the canonical aspartate transaminase reaction rate and connects alanine biosynthesis to glutamate via aspartate. This may explain the lack of any glutamate – pyruvate transaminase activity in R. leguminosarum, which is common in eukaryotic and many prokaryotic genomes. However, the aspartate to pyruvate transaminase reaction is not needed for N2 fixation in legume nodules. Consequently, we show that aspartate degradation is required for N2-fixation, rather than biosynthetic transamination to form an amino acid. Consequently, the enzyme aspartase, that catalyses the breakdown of aspartate to fumarate and ammonia, suppressed an AatA mutant and restored N2-fixation in pea nodules

Nodulation and nitrogen fixation in Medicago truncatula strongly alters the abundance of its root microbiota and subtly affects its structure.

Funder: University of Massachusetts Amherst; Id: http://dx.doi.org/10.13039/100008975The plant common symbiosis signalling (SYM) pathway has shared function between interactions with rhizobia and arbuscular mycorrhizal fungi, the two most important symbiotic interactions between plants and microorganisms that are crucial in plant and agricultural yields. Here, we determine the role of the plant SYM pathway in the structure and abundance of the microbiota in the model legume Medicago truncatula and whether this is controlled by the nitrogen or phosphorus status of the plant. We show that SYM mutants (dmi3) differ substantially from the wild type (WT) in the absolute abundance of the root microbiota, especially under nitrogen limitation. Changes in the structure of the microbiota were less pronounced and depended on both plant genotype and nutrient status. Thus, the SYM pathway has a major impact on microbial abundance in M. truncatula and also subtly alters the composition of the microbiota

Use of the Bruker MALDI Biotyper for identification of molds in the clinical mycology laboratory

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is increasingly used for the identification of bacteria and fungi in the diagnostic laboratory. We evaluated the mold database of Bruker Daltonik (Bremen, Germany), the Filamentous Fungi Library 1.0. First, we studied 83 phenotypically and molecularly well-characterized, nondermatophyte, nondematiaceous molds from a clinical strain collection. Using the manufacturer-recommended interpretation criteria, genus and species identification rates were 78.3% and 54.2%, respectively. Reducing the species cutoff from 2.0 to 1.7 significantly increased species identification to 71.1% without increasing misidentifications. In a subsequent prospective study, 200 consecutive clinical mold isolates were identified by the MALDI Biotyper and our conventional identification algorithm. Discrepancies were resolved by ribosomal DNA (rDNA) internal transcribed spacer region sequence analysis. For the MALDI Biotyper, genus and species identification rates were 83.5% and 79.0%, respectively, when using a species cutoff of 1.7. Not identified were 16.5% of the isolates. Concordant genus and species assignments of MALDI-TOF MS and the conventional identification algorithm were observed for 98.2% and 64.2% of the isolates, respectively. Four erroneous species assignments were observed using the MALDI Biotyper. The MALDI Biotyper seems highly reliable for the identification of molds when using the Filamentous Fungi Library 1.0 and a species cutoff of 1.7. However, expansion of the database is required to reduce the number of nonidentified isolates

Bradyrhizobium diazoefficiens Requires Chemical Chaperones To Cope with Osmotic Stress during Soybean Infection

When engaging in symbiosis with legume hosts, rhizobia are confronted with environmental changes, including nutrient availability and stress exposure. Genetic circuits allow responding to these environmental stimuli to optimize physiological adaptations during the switch from the free-living to the symbiotic life style. A pivotal regulatory system of the nitrogen-fixing soybean endosymbiont Bradyrhizobium diazoefficiens for efficient symbiosis is the general stress response (GSR), which relies on the alternative sigma factor σEcfG. However, the GSR-controlled process required for symbiosis has not been identified. Here, we demonstrate that biosynthesis of trehalose is under GSR control, and mutants lacking the respective biosynthetic genes otsA and/or otsB phenocopy GSR-deficient mutants under symbiotic and selected free-living stress conditions. The role of trehalose as a cytoplasmic chemical chaperone and stress protectant can be functionally replaced in an otsA or otsB mutant by introducing heterologous genetic pathways for biosynthesis of the chemically unrelated compatible solutes glycine betaine and (hydroxy)ectoine. Alternatively, uptake of exogenously provided trehalose also restores efficient symbiosis and tolerance to hyperosmotic and hyperionic stress of otsA mutants. Hence, elevated cytoplasmic trehalose levels resulting from GSR-controlled biosynthesis are crucial for B. diazoefficiens cells to overcome adverse conditions during early stages of host infection and ensure synchronization with root nodule development. IMPORTANCE The Bradyrhizobium-soybean symbiosis is of great agricultural significance and serves as a model system for fundamental research in bacterium-plant interactions. While detailed molecular insight is available about mutual recognition and early nodule organogenesis, our understanding of the host-imposed conditions and the physiology of infecting rhizobia during the transition from a free-living state in the rhizosphere to endosymbiotic bacteroids is currently limited. In this study, we show that the requirement of the rhizobial general stress response (GSR) during host infection is attributable to GSR-controlled biosynthesis of trehalose. Specifically, trehalose is crucial for an efficient symbiosis by acting as a chemical chaperone to protect rhizobia from osmostress during host infection.ISSN:2150-7511ISSN:2161-212

Salt- and Osmo-Responsive Sensor Histidine Kinases Activate the Bradyrhizobium diazoefficiens General Stress Response to Initiate Functional Symbiosis

The general stress response (GSR) enables bacteria to sense and overcome a variety of environmental stresses. In alphaproteobacteria, stress-perceiving histidine kinases of the HWE and HisKA_2 families trigger a signaling cascade that leads to phosphorylation of the response regulator PhyR and, consequently, to activation of the GSR σ factor σEcfG. In the nitrogen-fixing bacterium Bradyrhizobium diazoefficiens, PhyR and σEcfG are crucial for tolerance against a variety of stresses under free-living conditions and also for efficient infection of its symbiotic host soybean. However, the molecular players involved in stress perception and activation of the GSR remained largely unknown. In this work, we first showed that a mutant variant of PhyR where the conserved phosphorylatable aspartate residue D194 was replaced by alanine (PhyRD194A) failed to complement the ΔphyR mutant in symbiosis, confirming that PhyR acts as a response regulator. To identify the PhyR-activating kinases in the nitrogen-fixing symbiont, we constructed in-frame deletion mutants lacking single, distinct combinations, or all of the 11 predicted HWE and HisKA_2 kinases, which we named HRXXN histidine kinases HhkA through HhkK. Phenotypic analysis of the mutants and complemented derivatives identified two functionally redundant kinases, HhkA and HhkE, that are required for nodulation competitiveness and during initiation of symbiosis. Using σEcfG-activity reporter strains, we further showed that both HhkA and HhkE activate the GSR in free-living cells exposed to salt and hyperosmotic stress. In conclusion, our data suggest that HhkA and HhkE trigger GSR activation in response to osmotically stressful conditions which B. diazoefficiens encounters during soybean host infection.ISSN:0894-028

Structural basis and mechanism for metallochaperone-assisted assembly of the CuA center in cytochrome oxidase

ISSN:2375-254