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.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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.

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

The mechanisms underlying the biogenesis of the structurally unique, binuclear Cu1.5+•Cu1.5+ redox center (CuA) on subunit II (CoxB) of cytochrome oxidases have been a long-standing mystery. Here, we reconstituted the CoxB•CuA center in vitro from apo-CoxB and the holo-forms of the copper transfer chaperones ScoI and PcuC. A previously unknown, highly stable ScoI•Cu2+•CoxB complex was shown to be rapidly formed as the first intermediate in the pathway. Moreover, our structural data revealed that PcuC has two copper-binding sites, one each for Cu1+ and Cu2+, and that only PcuC•Cu1+•Cu2+ can release CoxB•Cu2+ from the ScoI•Cu2+•CoxB complex. The CoxB•CuA center was then formed quantitatively by transfer of Cu1+ from a second equivalent of PcuC•Cu1+•Cu2+ to CoxB•Cu2+. This metalation pathway is consistent with all available in vivo data and identifies the sources of the Cu ions required for CuA center formation and the order of their delivery to CoxB.

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 cbb 3 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 fixK 2, 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.

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.

Requirements for Efficient Thiosulfate Oxidation in Bradyrhizobium diazoefficiens.

One of the many disparate lifestyles of Bradyrhizobium diazoefficiens is chemolithotrophic growth with thiosulfate as an electron donor for respiration. The employed carbon source may be CO2 (autotrophy) or an organic compound such as succinate (mixotrophy). Here, we discovered three new facets of this capacity: (i) When thiosulfate and succinate were consumed concomitantly in conditions of mixotrophy, even a high molar excess of succinate did not exert efficient catabolite repression over the use of thiosulfate. (ii) Using appropriate cytochrome mutants, we found that electrons derived from thiosulfate during chemolithoautotrophic growth are preferentially channeled via cytochrome c550 to the aa3-type heme-copper cytochrome oxidase. (iii) Three genetic regulators were identified to act at least partially in the expression control of genes for chemolithoautotrophic thiosulfate oxidation: RegR and CbbR as activators, and SoxR as a repressor.

Metabolomic Profiling of Bradyrhizobium diazoefficiens-Induced Root Nodules Reveals Both Host Plant-Specific and Developmental Signatures.

Bradyrhizobium diazoefficiens is a nitrogen-fixing endosymbiont, which can grow inside root-nodule cells of the agriculturally important soybean and other host plants. Our previous studies described B. diazoefficiens host-specific global expression changes occurring during legume infection at the transcript and protein level. In order to further characterize nodule metabolism, we here determine by flow injection-time-of-flight mass spectrometry analysis the metabolome of (i) nodules and roots from four different B. diazoefficiens host plants; (ii) soybean nodules harvested at different time points during nodule development; and (iii) soybean nodules infected by two strains mutated in key genes for nitrogen fixation, respectively. Ribose (soybean), tartaric acid (mungbean), hydroxybutanoyloxybutanoate (siratro) and catechol (cowpea) were among the metabolites found to be specifically elevated in one of the respective host plants. While the level of C4-dicarboxylic acids decreased during soybean nodule development, we observed an accumulation of trehalose-phosphate at 21 days post infection (dpi). Moreover, nodules from non-nitrogen-fixing bacteroids (nifA and nifH mutants) showed specific metabolic alterations; these were also supported by independent transcriptomics data. The alterations included signs of nitrogen limitation in both mutants, and an increased level of a phytoalexin in nodules induced by the nifA mutant, suggesting that the tissue of these nodules exhibits defense and stress reactions.

Genome-wide transcription start site mapping of Bradyrhizobium japonicum grown free-living or in symbiosis - a rich resource to identify new transcripts, proteins and to study gene regulation.

BACKGROUND: Differential RNA-sequencing (dRNA-seq) is indispensable for determination of primary transcriptomes. However, using dRNA-seq data to map transcriptional start sites (TSSs) and promoters genome-wide is a bioinformatics challenge. We performed dRNA-seq of Bradyrhizobium japonicum USDA 110, the nitrogen-fixing symbiont of soybean, and developed algorithms to map TSSs and promoters. RESULTS: A specialized machine learning procedure for TSS recognition allowed us to map 15,923 TSSs: 14,360 in free-living bacteria, 4329 in symbiosis with soybean and 2766 in both conditions. Further, we provide proteomic evidence for 4090 proteins, among them 107 proteins corresponding to new genes and 178 proteins with N-termini different from the existing annotation (72 and 109 of them with TSS support, respectively). Guided by proteomics evidence, previously identified TSSs and TSSs experimentally validated here, we assign a score threshold to flag 14 % of the mapped TSSs as a class of lower confidence. However, this class of lower confidence contains valid TSSs of low-abundant transcripts. Moreover, we developed a de novo algorithm to identify promoter motifs upstream of mapped TSSs, which is publicly available, and found motifs mainly used in symbiosis (similar to RpoN-dependent promoters) or under both conditions (similar to RpoD-dependent promoters). Mapped TSSs and putative promoters, proteomic evidence and updated gene annotation were combined into an annotation file. CONCLUSIONS: The genome-wide TSS and promoter maps along with the extended genome annotation of B. japonicum represent a valuable resource for future systems biology studies and for detailed analyses of individual non-coding transcripts and ORFs. Our data will also provide new insights into bacterial gene regulation during the agriculturally important symbiosis between rhizobia and legumes.

Versatile Vectors for Efficient Mutagenesis of Bradyrhizobium diazoefficiens and Other Alphaproteobacteria.

UNLABELLED: Analysis of bacterial gene function commonly relies on gene disruption or replacement followed by phenotypic characterization of the resulting mutant strains. Deletion or replacement of targeted regions is commonly achieved via two homologous recombination (HR) events between the bacterial genome and a nonreplicating plasmid carrying DNA fragments flanking the region to be deleted. The counterselection of clones that have integrated the entire plasmid in their genome via a single HR event is crucial in this procedure. Various genetic tools and well-established protocols are available for this type of mutagenesis in model bacteria; however, these methods are not always efficiently applicable in less established systems. Here we describe the construction and application of versatile plasmid vectors pREDSIX and pTETSIX for marker replacement and markerless mutagenesis, respectively. Apart from an array of restriction sites optimized for cloning of GC-rich DNA fragments, the vector backbone contains a constitutively expressed gene for mCherry, enabling the rapid identification of clones originating from single or double HR events by fluorescence-assisted cell sorting (FACS). In parallel, we constructed a series of plasmids from which gene cassettes providing resistance against gentamicin, kanamycin, hygromycin B, streptomycin and spectinomycin, or tetracycline were excised for use with pREDSIX-based marker replacement mutagenesis. In proof-of-concept mutagenesis experiments, we demonstrated the potential for the use of the developed tools for gene deletion mutagenesis in the nitrogen-fixing soybean symbiont Bradyrhizobium diazoefficiens(formerly Bradyrhizobium japonicum) and three additional members of the alphaproteobacteria. IMPORTANCE: Mutation and phenotypic analysis are essential to the study of gene function. Efficient mutagenesis protocols and tools are available for many bacterial species, including various model organisms; however, genetic analysis of less-well-characterized organisms is often impaired by the lack of efficient methods. Here we describe a set of novel genetic tools for facilitated mutagenesis of the nitrogen-fixing soybean symbiont Bradyrhizobium diazoefficiens and related alphaproteobacteria. We demonstrated their usefulness by generating several mutant strains lacking defined genes. Isolation of both antibiotic resistance gene-containing and markerless deletion mutants is greatly facilitated because undesired clones which contain the entire mutagenic plasmid integrated in the genome can be identified on the basis of their fluorescent phenotype derived from them Cherrygene carried by the vector backbone. The possibility to generate markerless mutants assists with the isolation of strains carrying multiple deletions, which can be crucial while studying functionally redundant genes.

Stable Fluorescent and Enzymatic Tagging of Bradyrhizobium diazoefficiens to Analyze Host-Plant Infection and Colonization.

Bradyrhizobium diazoefficiens USDA 110 (formerly named Bradyrhizobium japonicum) can fix dinitrogen when living as an endosymbiont in root nodules of soybean and some other legumes. Formation of a functional symbiosis relies on a defined developmental program mediated by controlled gene expression in both symbiotic partners. In contrast to other well-studied Rhizobium-legume model systems that have been thoroughly examined by means of genetically tagged strains, analysis of B. diazoefficiens host infection has been impaired due to the lack of suitable tagging systems. Here, we describe the construction of B. diazoefficiens strains constitutively expressing single-copy genes for fluorescent proteins (eBFP2, mTurquoise2, GFP+, sYFP2, mCherry, HcRed) and enzymes (GusA, LacZ). For stable inheritance, the constructs were recombined into the chromosome. Effectiveness and versatility of the tagged strains was demonstrated in plant infection assays. (i) The infection process was followed from root-hair attachment to colonization of nodule cells with epifluorescent microscopy. (ii) Monitoring mixed infections with two strains producing different fluorescent proteins allowed rapid analysis of nodule occupancy and revealed that the majority of nodules contained clonal populations. (iii) Microscopic analysis of nodules induced by fluorescent strains provided evidence for host-dependent control of B. diazoefficiens bacteroid morphology in nodules of Aeschynomene afraspera and Arachis hypogaea (peanut), as deduced from their altered morphology compared with bacteroids in soybean nodules.

The general stress response in Alphaproteobacteria.

The general stress response (GSR) is a widely conserved response that allows bacteria to cope with a multitude of stressful conditions. In the past years the PhyR-NepR-σ(EcfG) cascade was identified as the core pathway regulating the GSR in Alphaproteobacteria, in which it also plays an important role in bacteria-host interactions. The regulatory system is composed of the extracytoplasmic function sigma factor σ(EcfG), its anti-sigma factor NepR (for negative regulator of the PhyR response), and the anti-sigma factor antagonist PhyR (phyllosphere regulator). The three proteins function via a partner-switching mechanism that is triggered by PhyR phosphorylation, termed 'sigma factor mimicry'. This review will cover core features of the pathway, its physiological role, and summarize recent advances towards understanding of the partner-switching mechanism and of the two-component signaling pathways controlling the GSR.

The global response regulator RegR controls expression of denitrification genes in Bradyrhizobium japonicum.

Bradyrhizobium japonicum RegSR regulatory proteins belong to the family of two-component regulatory systems, and orthologs are present in many Proteobacteria where they globally control gene expression mostly in a redox-responsive manner. In this work, we have performed a transcriptional profiling of wild-type and regR mutant cells grown under anoxic denitrifying conditions. The comparative analyses of wild-type and regR strains revealed that almost 620 genes induced in the wild type under denitrifying conditions were regulated (directly or indirectly) by RegR, pointing out the important role of this protein as a global regulator of denitrification. Genes controlled by RegR included nor and nos structural genes encoding nitric oxide and nitrous oxide reductase, respectively, genes encoding electron transport proteins such as cycA (blr7544) or cy2 (bll2388), and genes involved in nitric oxide detoxification (blr2806-09) and copper homeostasis (copCAB), as well as two regulatory genes (bll3466, bll4130). Purified RegR interacted with the promoters of norC (blr3214), nosR (blr0314), a fixK-like gene (bll3466), and bll4130, which encodes a LysR-type regulator. By using fluorescently labeled oligonucleotide extension (FLOE), we were able to identify two transcriptional start sites located at about 35 (P1) and 22 (P2) bp upstream of the putative translational start codon of norC. P1 matched with the previously mapped 5'end of norC mRNA which we demonstrate in this work to be under FixK2 control. P2 is a start site modulated by RegR and specific for anoxic conditions. Moreover, qRT-PCR experiments, expression studies with a norC-lacZ fusion, and heme c-staining analyses revealed that anoxia and nitrate are required for RegR-dependent induction of nor genes, and that this control is independent of the sensor protein RegS.

Thioredoxin-like protein TlpA from Bradyrhizobium japonicum is a reductant for the copper metallochaperone ScoI.

TlpA and ScoI of Bradyrhizobium japonicum are membrane-anchored thioredoxin-like proteins oriented towards the periplasm. TlpA is a protein-disulfide reductase. ScoI is a copper chaperone for cytochrome oxidase biogenesis. TlpA with its negative redox potential (E(o') -256 mV) was shown here to reduce oxidized ScoI, for which we determined a less negative E(o') (-160 mV). The fast forward reaction (rate constant 9.4×10(4) M(-1) s(-1)) was typical for physiologically relevant disulfide exchange reactions. A transient TlpA-ScoI heterodisulfide formed between Cys107 of TlpA's active site (C(107)XXC(110)) and Cys78 of ScoI's copper-binding site (C(74)XXXC(78)). We conclude that TlpA recycles ScoI to the dithiol form prior to metallation.

Copper starvation-inducible protein for cytochrome oxidase biogenesis in Bradyrhizobium japonicum.

Microarray analysis of Bradyrhizobium japonicum grown under copper limitation uncovered five genes named pcuABCDE, which are co-transcribed and co-regulated as an operon. The predicted gene products are periplasmic proteins (PcuA, PcuC, and PcuD), a TonB-dependent outer membrane receptor (PcuB), and a cytoplasmic membrane-integral protein (PcuE). Homologs of PcuC and PcuE had been discovered in other bacteria, namely PCu(A)C and YcnJ, where they play a role in cytochrome oxidase biogenesis and copper transport, respectively. Deletion of the pcuABCDE operon led to a pleiotropic phenotype, including defects in the aa(3)-type cytochrome oxidase, symbiotic nitrogen fixation, and anoxic nitrate respiration. Complementation analyses revealed that, under our assay conditions, the tested functions depended only on the pcuC gene and not on pcuA, pcuB, pcuD, or pcuE. The B. japonicum genome harbors a second pcuC-like gene (blr7088), which, however, did not functionally replace the mutated pcuC. The PcuC protein was overexpressed in Escherichia coli, purified to homogeneity, and shown to bind Cu(I) with high affinity in a 1:1 stoichiometry. The replacement of His(79), Met(90), His(113), and Met(115) by alanine perturbed copper binding. This corroborates the previously purported role of this protein as a periplasmic copper chaperone for the formation of the Cu(A) center on the aa(3)-type cytochrome oxidase. In addition, we provide evidence that PcuC and the copper chaperone ScoI are important for the symbiotically essential, Cu(A)-free cbb(3)-type cytochrome oxidase specifically in endosymbiotic bacteroids of soybean root nodules, which could explain the symbiosis-defective phenotype of the pcuC and scoI mutants.

Reactive oxygen species-inducible ECF σ factors of Bradyrhizobium japonicum.

Extracytoplasmic function (ECF) σ factors control the transcription of genes involved in different cellular functions, such as stress responses, metal homeostasis, virulence-related traits, and cell envelope structure. The genome of Bradyrhizobium japonicum, the nitrogen-fixing soybean endosymbiont, encodes 17 putative ECF σ factors belonging to nine different ECF σ factor families. The genes for two of them, ecfQ (bll1028) and ecfF (blr3038), are highly induced in response to the reactive oxygen species hydrogen peroxide (H(2)O(2)) and singlet oxygen ((1)O(2)). The ecfF gene is followed by the predicted anti-σ factor gene osrA (blr3039). Mutants lacking EcfQ, EcfF plus OsrA, OsrA alone, or both σ factors plus OsrA were phenotypically characterized. While the symbiotic properties of all mutants were indistinguishable from the wild type, they showed increased sensitivity to singlet oxygen under free-living conditions. Possible target genes of EcfQ and EcfF were determined by microarray analyses, and candidate genes were compared with the H(2)O(2)-responsive regulon. These experiments disclosed that the two σ factors control rather small and, for the most part, distinct sets of genes, with about half of the genes representing 13% of the members of H(2)O(2)-responsive regulon. To get more insight into transcriptional regulation of both σ factors, the 5' ends of ecfQ and ecfF mRNA were determined. The presence of conserved sequence motifs in the promoter region of ecfQ and genes encoding EcfQ-like σ factors in related α-proteobacteria suggests regulation via a yet unknown transcription factor. By contrast, we have evidence that ecfF is autoregulated by transcription from an EcfF-dependent consensus promoter, and its product is negatively regulated via protein-protein interaction with OsrA. Conserved cysteine residues 129 and 179 of OsrA are required for normal function of OsrA. Cysteine 179 is essential for release of EcfF from an EcfF-OsrA complex upon H(2)O(2) stress while cysteine 129 is possibly needed for EcfF-OsrA interaction.

Genome-wide transcription analysis of Escherichia coli in response to extremely low-frequency magnetic fields.

The widespread use of electricity raises the question of whether or not 50 Hz (power line frequency in Europe) magnetic fields (MFs) affect organisms. We investigated the transcription of Escherichia coli K-12 MG1655 in response to extremely low-frequency (ELF) MFs. Fields generated by three signal types (sinusoidal continuous, sinusoidal intermittent, and power line intermittent; all at 50 Hz, 1 mT) were applied and gene expression was monitored at the transcript level using an Affymetrix whole-genome microarray. Bacterial cells were grown continuously in a chemostat (dilution rate D = 0.4 h(-1)) fed with glucose-limited minimal medium and exposed to 50 Hz MFs with a homogenous flux density of 1 mT. For all three types of MFs investigated, neither bacterial growth (determined using optical density) nor culturable counts were affected. Likewise, no statistically significant change (fold-change > 2, P ≤ 0.01) in the expression of 4,358 genes and 714 intergenic regions represented on the gene chip was detected after MF exposure for 2.5 h (1.4 generations) or 15 h (8.7 generations). Moreover, short-term exposure (8 min) to the sinusoidal continuous and power line intermittent signal neither affected bacterial growth nor showed evidence for reliable changes in transcription. In conclusion, our experiments did not indicate that the different tested MFs (50 Hz, 1 mT) affected the transcription of E. coli.

Small RNAs of the Bradyrhizobium/Rhodopseudomonas lineage and their analysis.

Small RNAs (sRNAs) play a pivotal role in bacterial gene regulation. However, the sRNAs of the vast majority of bacteria with sequenced genomes still remain unknown since sRNA genes are usually difficult to recognize and thus not annotated. Here, expression of seven sRNAs (BjrC2a, BjrC2b, BjrC2c, BjrC68, BjrC80, BjrC174 and BjrC1505) predicted by genome comparison of Bradyrhizobium and Rhodopseudomonas members, was verified by RNA gel blot hybridization, microarray and deep sequencing analyses of RNA from the soybean symbiont Bradyrhizobium japonicum USDA 110. BjrC2a, BjrC2b and BjrC2c belong to the RNA family RF00519, while the other sRNAs are novel. For some of the sRNAs we observed expression differences between free-living bacteria and bacteroids in root nodules. The amount of BjrC1505 was decreased in nodules. By contrast, the amount of BjrC2a, BjrC68, BjrC80, BjrC174 and the previously described 6S RNA was increased in nodules, and accumulation of truncated forms of these sRNAs was observed. Comparative genomics and deep sequencing suggest that BjrC2a is an antisense RNA regulating the expression of inositol-monophosphatase. The analyzed sRNAs show a different degree of conservation in Rhizobiales, and expression of homologs of BjrC2, BjrC68, BjrC1505, and 6S RNA was confirmed in the free-living purple bacterium Rhodopseudomonas palustris 5D.

Lack of glyphosate resistance gene transfer from Roundup Ready soybean to Bradyrhizobium japonicum under field and laboratory conditions.

A field study was conducted at the Russell E. Larson Agricultural Research Center to determine the effect of transgenic glyphosate-resistant soybean in combination with herbicide (Roundup) application on its endosymbiont Bradyrhizobium japonicum. DNA of bacteroids from isolated nodules was analysed for the presence of the transgenic 5-enolpyruvylshikimate-3-phosphate synthase (CP4-EPSPS) DNA sequence using polymerase chain reaction (PCR). To further assess the likelihood that the EPSPS gene may be transferred from the Roundup Ready (RR) soybean to B. japonicum, we have examined the natural transformation efficiency of B. japonicum strain 110spc4. Analyses of nodules showed the presence of the transgenic EPSPS DNA sequence. In bacteroids that were isolated from nodules of transgenic soybean plants and then cultivated in the presence of glyphosate this sequence could not be detected. This indicates that no stable horizontal gene transfer (HGT) of the EPSPS gene had occurred under field conditions. Under laboratory conditions, no natural transformation was detected in B. japonicum strain 110spc4 in the presence of various amounts of recombinant plasmid DNA. Our results indicate that no natural competence state exists in B. japonicum 110spc4. Results from field and laboratory studies indicate the lack of functional transfer of the CP4-EPSPS gene from glyphosate-tolerant soybean treated with glyphosate to root-associated B. japonicum.

Host-specific symbiotic requirement of BdeAB, a RegR-controlled RND-type efflux system in Bradyrhizobium japonicum.

Multidrug efflux systems not only cause resistance against antibiotics and toxic compounds but also mediate successful host colonization by certain plant-associated bacteria. The genome of the nitrogen-fixing soybean symbiont Bradyrhizobium japonicum encodes 24 members of the family of resistance/nodulation/cell division (RND) multidrug efflux systems, of which BdeAB is genetically controlled by the RegSR two-component regulatory system. Phylogenetic analysis of the membrane components of these 24 RND-type transporters revealed that BdeB is more closely related to functionally characterized orthologs in other bacteria, including those associated with plants, than to any of the other 23 paralogs in B. japonicum. A mutant with a deletion of the bdeAB genes was more susceptible to inhibition by the aminoglycosides kanamycin and gentamicin than the wild type, and had a strongly decreased symbiotic nitrogen-fixation activity on soybean, but not on the alternative host plants mungbean and cowpea, and only very marginally on siratro. The host-specific role of a multidrug efflux pump is a novel feature in the rhizobia-legume symbioses. Consistent with the RegSR dependency of bdeAB, a B. japonicum regR mutant was found to have a greater sensitivity against the two tested antibiotics and a symbiotic defect that is most pronounced for soybean.

Disparate pathways for the biogenesis of cytochrome oxidases in Bradyrhizobium japonicum.

This work addresses the biogenesis of heme-copper terminal oxidases in Bradyrhizobium japonicum, the nitrogen-fixing root nodule symbiont of soybean. B. japonicum has four quinol oxidases and four cytochrome oxidases. The latter include the aa(3)- and cbb(3)-type oxidases. Although both have a Cu(B) center in subunit I, the subunit II proteins differ in having either a Cu(A) center (in aa(3)) or a covalently bound heme c (in cbb(3)). Two biogenesis factors were genetically studied here, the periplasmically exposed CoxG and ScoI proteins, which are the respective homologs of the mitochondrial copper-trafficking chaperones Cox11 and Sco1 for the formation of the Cu(B) center in subunit I and the Cu(A) center in subunit II of cytochrome aa(3). We could demonstrate copper binding to ScoI in vitro, a process for which the thiols of cysteine residues 74 and 78 in the ScoI polypeptide were shown to be essential. Knock-out mutations in the B. japonicum coxG and scoI genes led to loss of cytochrome aa(3) assembly and activity in the cytoplasmic membrane, whereas the cbb(3)-type cytochrome oxidase apparently remained unaffected. This suggests that subunit I of the cbb(3)-type oxidase obtains its copper cofactor via a different pathway than cytochrome aa(3). In contrast to the coxG mutation, the scoI mutation caused a decreased symbiotic nitrogen fixation activity. We hypothesize that a periplasmic B. japonicum protein other than any of the identified Cu(A) proteins depends on ScoI and is required for an effective symbiosis.