The Bradyrhizobium japonicum phoB gene is required for phosphate-limited growth but not for symbiotic nitrogen fixation

We identified by cloning and DNA sequence analysis the phosphate regulatory gene phoB of Bradyrhizobium japonicum. The deduced gene product displayed pronounced similarity to the PhoB protein of Sinorhizobium meliloti (71.4% identical amino acids), Escherichia coli (50.2%) and other bacterial species. Insertion of a kanamycin resistance cassette into phoB led to impaired growth of the B. japonicum mutant in media containing approximately 25 μM phosphate or less. A standard plant infection test using wild-type and phoB-defective B. japonicum strains showed that the phoB mutation had no effect on the symbiotic properties of B. japonicum with its soybean host plan

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 soybea

Dissection of the Bradyrhizobium japonicum NifA+σ54 regulon, and identification of a ferredoxin gene ( fdxN ) for symbiotic nitrogen fixation

Hierarchically organized regulatory proteins form a complex network for expression control of symbiotic and accessory genes in the nitrogen-fixing soybean symbiont Bradyrhizobium japonicum. A genome-wide survey of regulatory interactions was made possible with the design of a custom-made gene chip. Here, we report the first use of the microarray in a comprehensive and complete characterization of the B. japonicum NifA+σ54 regulon which forms an important node in the entire network. Comparative transcript profiles of anaerobically grown wild-type, nifA, and rpoN 1/2 mutant cells were complemented with a position-specific frequency matrix-based search for NifA- and σ54-binding sites plus a simple operon definition. One of the newly identified NifA+σ54-dependent genes, fdxN, encodes a ferredoxin required for efficient symbiotic nitrogen fixation, which makes it a candidate for being a direct electron donor to nitrogenase. The fdxN gene has an unconventional, albeit functional σ54 promoter with the dinucleotide GA instead of the consensus GC motif at position −12. A GC-containing mutant promoter and the atypical GA-containing promoter of the wild type were disparately activated. Expression analyses were also carried out with two other NifA+σ54 targets (ectC; ahpC). Incidentally, the tiling-like design of the microarray has helped to arrive at completely revised annotations of the ectC- and ahpC-upstream DNA regions, which are now compatible with promoter locations. Taken together, the approaches used here led to a substantial expansion of the NifA+σ54 regulon size, culminating in a total of 65 genes for nitrogen fixation and diverse other processe

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 cy 2 (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 9 end of norC mRNA which we demonstrate in this work to be under FixK 2 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 Reg

Threads of memory: Reviving the ornament of a dead child at the Neolithic village of Ba`ja (Jordan)

In 2018, a well-constructed cist-type grave was discovered at Ba`ja, a Neolithic village (7,400–6,800 BCE) in Southern Jordan. Underneath multiple grave layers, an 8-year-old child was buried in a fetal position. Over 2,500 beads were found on the chest and neck, along with a double perforated stone pendant and a delicately engraved mother-of-pearl ring discovered among the concentration of beads. The first was found behind the neck, and the second on the chest. The meticulous documentation of the bead distribution indicated that the assemblage was a composite ornament that had gradually collapsed, partly due to the burying position. Our aim was to challenge time degradation and to reimagine the initial composition in order to best explore the significance of this symbolic category of material culture, not as mere group of beads, but as an ornamental creation with further aesthetic, artisanal and socioeconomic implications. The reconstruction results exceeded our expectations as it revealed an imposing multi-row necklace of complex structure and attractive design. Through multiple lines of evidence, we suggest that the necklace was created at Ba`ja, although significant parts of beads were made from exotic shells and stones, including fossil amber, an unprecedented material never attested before for this period. The retrieval of such an ornament from life and its attribution to a young dead child highlights the significant social status of this individual. Beyond the symbolic functions related to identity, the necklace is believed to have played a key role in performing the inhumation rituals, understood as a public event gathering families, relatives, and people from other villages. In this sense, the necklace is not seen as belonging completely to the realm of death but rather to the world of the living, materializing a collective memory and shared moments of emotions and social cohesion

Planet Hunters. V. A Confirmed Jupiter-Size Planet in the Habitable Zone and 42 Planet Candidates from the Kepler Archive Data

We report the latest Planet Hunter results, including PH2 b, a Jupiter-size (R_PL = 10.12 \pm 0.56 R_E) planet orbiting in the habitable zone of a solar-type star. PH2 b was elevated from candidate status when a series of false positive tests yielded a 99.9% confidence level that transit events detected around the star KIC 12735740 had a planetary origin. Planet Hunter volunteers have also discovered 42 new planet candidates in the Kepler public archive data, of which 33 have at least three transits recorded. Most of these transit candidates have orbital periods longer than 100 days and 20 are potentially located in the habitable zones of their host stars. Nine candidates were detected with only two transit events and the prospective periods are longer than 400 days. The photometric models suggest that these objects have radii that range between Neptune to Jupiter. These detections nearly double the number of gas giant planet candidates orbiting at habitable zone distances. We conducted spectroscopic observations for nine of the brighter targets to improve the stellar parameters and we obtained adaptive optics imaging for four of the stars to search for blended background or foreground stars that could confuse our photometric modeling. We present an iterative analysis method to derive the stellar and planet properties and uncertainties by combining the available spectroscopic parameters, stellar evolution models, and transiting light curve parameters, weighted by the measurement errors. Planet Hunters is a citizen science project that crowd-sources the assessment of NASA Kepler light curves. The discovery of these 43 planet candidates demonstrates the success of citizen scientists at identifying planet candidates, even in longer period orbits with only two or three transit events.Comment: 35 pages, 11 figures, 6 tables, accepted and published on ApJ ApJ, 776, 1