This study focuses on the function of the gene praR that encodes a putative transcription factor in Azorhizobium caulinodans ORS571, a microsymbiont of Sesbania rostrata. The praR gene is a homolog of the phrR gene of Sinorhizobium medicae WSM419, and the praR and phrR homologs are distributed throughout the class Alphaproteobacteria. The growth and nitrogen fixation activity of an A. caulinodans praR deletion mutant in the free-living state were not significantly different from those of the wild-type strain. However, the stem nodules formed by the praR mutant showed lower nitrogen fixation activity than the wild-type stem nodules. Microscopy revealed that infected host cells with an oval or elongated shape were observed at early stages in the nodules formed by the praR mutant, but these infected cells gradually fell into two types. One maintained an oval or elongated shape, but the vacuoles in these cells gradually enlarged and the bacteria gradually disappeared. The other cells were shrunken with bacteria remaining inside. Microarrays revealed that genes homologous to the reb genes of Caedibacter taeniospiralis were highly expressed in the praR mutant. Furthermore, the stem nodules formed by an A. caulinodans mutant with a deletion of praR and reb-homologous genes showed high nitrogen fixation activity, comparable to that of the wild-type stem nodules, and were filled with oval or elongated host cells. These results suggest that PraR controls the expression of the reb-homologous genes and that high expression of reb-homologous genes causes aberrance in A. caulinodans-S. rostrata symbiosis.
The whole-genome sequence of the endosymbiotic bacterium Azorhizobium caulinodans ORS571, which forms nitrogen-fixing nodules on the stems and roots of Sesbania rostrata, was recently determined. The sizes of the genome and symbiosis island are 5.4 Mb and 86.7 kb, respectively, and these sizes are the smallest among the sequenced rhizobia. In the present study, a whole-genome microarray of A. caulinodans was constructed, and transcriptomic analyses were performed on free-living cells grown in rich and minimal media and in bacteroids isolated from stem nodules. Transcriptional profiling showed that the genes involved in sulfur uptake and metabolism, acetone metabolism, and the biosynthesis of exopolysaccharide were highly expressed in bacteroids compared to the expression levels in free-living cells. Some mutants having Tn5 transposons within these genes with increased expression were obtained as nodule-deficient mutants in our previous study. A transcriptomic analysis was also performed on free-living cells grown in minimal medium supplemented with a flavonoid, naringenin, which is one of the most efficient inducers of A. caulinodans nod genes. Only 18 genes exhibited increased expression by the addition of naringenin, suggesting that the regulatory mechanism responding to the flavonoid could be simple in A. caulinodans. The combination of our genome-wide transcriptional profiling and our previous genome-wide mutagenesis study has revealed new aspects of nodule formation and maintenance.The symbiosis between rhizobia and legumes results in the formation of nitrogen-fixing nodules. The symbiotic interaction begins with the induction of bacterial nod genes by flavonoids secreted from the plant roots (8). The nod genes encode proteins that synthesize the nodulation (Nod) factor, which initiates many developmental changes, such as root hair curling and root cell division required for the formation of the nodule primordium in the host plant early during the nodulation process (8,24,50). Bacteria are entrapped in the curled root hair and subsequently infect the root hair through infection threads made of the plant cell wall. Upon release from the infection threads, bacteria invade the plant cell cytoplasm, where they differentiate into bacteroids and provide ammonium to the host plant by reducing atmospheric dinitrogen in exchange for carbon and amino acid compounds (16,49,53). It has been deduced that multiple stages exist in the establishment of nitrogen-fixing symbiosis. To identify novel genes involved in various stages of symbiosis, transcriptomic studies based on complete genome sequences were performed using Sinorhizobium (1, 2, 5, 9), Mesorhizobium (71), and Bradyrhizobium (10,42,54).Azorhizobium caulinodans ORS571 is a microsymbiont of Sesbania rostrata (18)(19)(20). Nitrogen-fixing nodules are formed by A. caulinodans on the stems as well as on the roots of S. rostrata. Stem nodules occur at the site of adventitious root primordia located on the stems after crack-entry invasion by A. caulinodans (70). During...
Bacteria have multiple K ϩ uptake systems. Escherichia coli, for example, has three types of K ϩ uptake systems, which include the low-K ϩ -inducible KdpFABC system and two constitutive systems, Trk (TrkAG and TrkAH) and Kup. Azorhizobium caulinodans ORS571, a rhizobium that forms nitrogen-fixing nodules on the stems and roots of Sesbania rostrata, also has three types of K ϩ uptake systems. Through phylogenetic analysis, we found that A. caulinodans has two genes homologous to trkG and trkH, designated trkI and trkJ. We also found that trkI is adjacent to trkA in the genome and these two genes are transcribed as an operon; however, trkJ is present at a distinct locus. Our results demonstrated that trkAI, trkJ, and kup were expressed in the wild-type stem nodules, whereas kdpFABC was not. Interestingly, Δkup and Δkup ΔkdpA mutants formed Fix -nodules, while the Δkup ΔtrkA ΔtrkI ΔtrkJ mutant formed Fix ϩ nodules, suggesting that with the additional deletion of Trk system genes in the Δkup mutant, Fix ϩ nodule phenotypes were recovered. kdpFABC of the Δkup ΔtrkJ mutant was expressed in stem nodules, but not in the free-living state, under high-K ϩ conditions. However, kdpFABC of the Δkup ΔtrkA ΔtrkI ΔtrkJ mutant was highly expressed even under high-K ϩ conditions. The cytoplasmic K ϩ levels in the Δkup ΔtrkA ΔtrkI mutant, which did not express kdp-FABC under high-K ϩ conditions, were markedly lower than those in the Δkup ΔtrkA ΔtrkI ΔtrkJ mutant. Taking all these results into consideration, we propose that TrkJ is involved in the repression of kdpFABC in response to high external K ϩ concentrations and that the TrkAI system is unable to function in stem nodules.IMPORTANCE K ϩ is a major cytoplasmic cation in prokaryotic and eukaryotic cells. Bacteria have multiple K ϩ uptake systems to control the cytoplasmic K ϩ levels. In many bacteria, the K ϩ uptake system KdpFABC is expressed under low-K ϩ conditions. For years, many researchers have argued over how bacteria sense K ϩ concentrations. Although KdpD of Escherichia coli is known to sense both cytoplasmic and extracellular K ϩ concentrations, the detailed mechanism of K ϩ sensing is still unclear. In this study, we propose that the transmembrane TrkJ protein of Azorhizobium caulinodans acts as a sensor for the extracellular K ϩ concentration and that high extracellular K ϩ concentrations repress the expression of KdpFABC via TrkJ. KEYWORDS potassium transport, rhizobium, symbiosisA zorhizobium caulinodans ORS571 is a microsymbiont of the water-tolerant tropical legume Sesbania rostrata (1-3), where it forms N 2 -fixing nodules on the stems and roots. A previous transposon mutagenesis study on the rhizobial factors involved in
As mobility products, such as smart phones, tablets and wearable devices, continue on the paths of both miniaturization and high performance, components and packaging are likewise driven to become smaller and thinner. In mobile devices, a thinner package is required, especially for the Application Processor and memory combination. The Package-on-Package structure can achieve module thinness by embedding a die (or dies) into a package and is thus attractive for reducing the height of an AP. The Die-Embedded and RDL structure has been in development as a package corresponding to POP structure. This structure has two characteristics of manufacturing method, based on the substrate manufacturing process. One is the RDL-first process, whereby the RDL is manufactured before die attachment. This process decreases the die loss from defects in the RDL formation. The other method is to manufacture the packages in panel form instead of wafer form. The key features of this structure are panel-form manufacturing and via connection between the substrate and the top-RDL. In IMAPS 2016, the Die Embedded and RDL structure on i-THOP®, which was used as the substrate was reported. The die-to-die inter connection was fabricated on i-THOP® for a 2.1D application. Both the die attachment and the RDL formation were successfully performed. The reliability tests proceeded. The results indicated that the i-THOP® with the Die Embedded and RDL structure can be applied to packaging of AP in mobile devices. The next challenge is to make a smaller package using the Die Embedded and RDL structure. To decrease the package area, a finer-pitch via connection that was less than the design rule of 200μm was evaluated. For reduction of package thickness, a thinner substrate, approximately 160μm thickness, was applied. The new test sample with these improvements was designed and proceeded to evaluation. The dimensions of the sample are 15 × 15 mm2 size and 340μm thickness (without solder ball), a 100μm-thick die was embedded and a one-layer of RDL was formed on the die. In this development, the warpage of the thin substrate after die attachment might be large, so there is an issue of package warpage and the handling of a thin substrate. In this report, results of low warpage structure design and development status of fine via connection formation are described.
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