The PTSNtr-KdpDE-KdpFABC Pathway Contributes to Low Potassium Stress Adaptation and Competitive Nodulation of Sinorhizobium fredii.

The rhizobium-legume symbiosis is essential for sustainable agriculture by reducing nitrogen fertilizer input, but its efficiency varies under fluctuating soil conditions and resources. The nitrogen-related phosphotransferase system (PTSNtr) consisting of PtsP, PtsO, and PtsN is required for optimal nodulation and nitrogen fixation efficiency of the broad-host-range Sinorhizobium fredii CCBAU45436 associated with diverse legumes, though the underlying mechanisms remain elusive. This work characterizes the PtsN-KdpDE-KdpFABC pathway that contributes to low potassium adaptation and competitive nodulation of CCBAU45436. Among three PtsN, PtsN1 is the major functional homolog. The unphosphorylated PtsN1 binds the sensory kinase KdpD through a non-canonical interaction with the GAF domain of KdpD, while the region covering HisKA-HATPase domains mediates the interaction of KdpD with the response regulator KdpE. KdpE directly activates the kdpFABC operon encoding the conserved high-affinity potassium uptake system. Disruption of this signaling pathway leads to reduced nodule number, nodule occupancy, and low potassium adaptation ability, but without notable effects on rhizoplane colonization. The induction of key nodulation genes NIN and ENOD40 in host roots during early symbiotic interactions is impaired when inoculating the kdpBC mutant that shows delayed nodulation. The nodulation defect of the kdpBC mutant can be rescued by supplying replete potassium. Potassium is actively consumed by both prokaryotes and eukaryotes, and components of the PTSNtr-KdpDE-KdpFABC pathway are widely conserved in bacteria, highlighting the global importance of this pathway in bacteria-host interactions. IMPORTANCE In all ecological niches, potassium is actively consumed by diverse prokaryotes and their interacting eukaryote hosts. It is only just emerging that potassium is a key player in host-pathogen interactions, and the role of potassium in mutualistic interactions remains largely unknown. This work is focused on the mutualistic symbiosis between rhizobia and legumes. We report that the nitrogen-related phosphotransferase system PTSNtr, the two-component system KdpDE, and the high-affinity potassium uptake system KdpFABC constitute a pathway that is important for low potassium adaptation and optimal nodulation of rhizobia. Given the widely conserved PTSNtr, KdpDE, and KdpFABC in bacteria and increasing knowledge on microbiome for various niches, the PTSNtr-KdpDE-KdpFABC pathway can be globally important in the biosphere.

Genetic Analysis Reveals the Essential Role of Nitrogen Phosphotransferase System Components in Sinorhizobium fredii CCBAU 45436 Symbioses with Soybean and Pigeonpea Plants.

The nitrogen phosphotransferase system (PTS(Ntr)) consists of EI(Ntr), NPr, and EIIA(Ntr). The active phosphate moiety derived from phosphoenolpyruvate is transferred through EI(Ntr) and NPr to EIIA(Ntr). Sinorhizobium fredii can establish a nitrogen-fixing symbiosis with the legume crops soybean (as determinate nodules) and pigeonpea (as indeterminate nodules). In this study, S. fredii strains with mutations in ptsP and ptsO (encoding EI(Ntr) and NPr, respectively) formed ineffective nodules on soybeans, while a strain with a ptsN mutation (encoding EIIA(Ntr)) was not defective in symbiosis with soybeans. Notable reductions in the numbers of bacteroids within each symbiosome and of poly-ß-hydroxybutyrate granules in bacteroids were observed in nodules infected by the ptsP or ptsO mutant strains but not in those infected with the ptsN mutant strain. However, these defects of the ptsP and ptsO mutant strains were recovered in ptsP ptsN and ptsO ptsN double-mutant strains, implying a negative role of unphosphorylated EIIA(Ntr) in symbiosis. Moreover, the symbiotic defect of the ptsP mutant was also recovered by expressing EI(Ntr) with or without the GAF domain, indicating that the putative glutamine-sensing domain GAF is dispensable in symbiotic interactions. The critical role of PTS(Ntr) in symbiosis was also observed when related PTS(Ntr) mutant strains of S. fredii were inoculated on pigeonpea plants. Furthermore, nodule occupancy and carbon utilization tests suggested that multiple outputs could be derived from components of PTS(Ntr) in addition to the negative role of unphosphorylated EIIA(Ntr).

Aldehyde dehydrogenase 1: a specific cancer stem cell marker for human colorectal carcinoma.

Since the identification of cancer stem cells (CSCs) a new understanding of tumor occurrence and development has evolved. According to the stem cell (SC) theory, colorectal carcinoma (CRC) SCs may be derived from mutations in normal intestinal cells. CSCs can be defined by their cell of origin (SCs or early progenitor cells). Thus, through a shared stem cell marker between CSCs and SCs, it is possible to investigate the association between its expression and the various clinicopathological features in patients with CRC. Aldehyde dehydrogenase 1 (ALDH1) is an appropriate marker. The present study was performed to examine the role of ALDH1 in CRC. Through indirect fluorescence antibody staining, the association between ALDH1 protein expression and various clinicopathological parameters was investigated. Furthermore, enzyme­linked immunosorbent assay (ELISA) was used to investigate the differing content of ALDH1 between CRC tissues and normal colorectal tissues. The results revealed that ALDH1 expression was markedly associated with tumor stage, Dukes' stage and the level of tumor cell differentiation. Using ELISA, it was demonstrated that there was a greater level of ALDH1 in CRC tissue than in normal colorectal tissue. Therefore, ALDH1 levels can be used as a useful parameter for pathological evaluation of tissue histology and to predict disease prognosis.

Inhibitory effect of schisandrin B on gastric cancer cells in vitro.

AIM: To investigate the inhibitory effect and possible mechanism of action of schisandrin B in SC-B on gastric cancer cells in vitro. METHODS: SC-B consisted of schisandrin B, aloe-emodin, and Astragalus polysaccharides. Exponentially growing human gastric cancer SGC-7901 cells were divided into six treatment groups: (1) control group (RPMI 1640 medium); (2) negative control group (2% DMSO); (3) positive control group (50 mg/L 5-Fluorouracil, 5-FU); (4) low-dose group (LSC, final concentration of schisandrin B, 25 mg/L); (5) moderate-dose group (MSC, final concentration of schisandrin B, 50 mg/L); (6) high-dose group (HSC, final concentration of schisandrin B, 100 mg/L). Follow-up was done at 12-48 h. An MTT (Methylthiazolyldiphenyl-tetrazolium bromide) assay was used to examine the inhibitory effect of SC-B on gastric cancer cells. The mitosis index was assessed using an inverted microscope. Flow cytometry was used to visualize the cell cycle. An RT-PCR (Reverse transcription-Polymerase chain reaction) -based assay was used to detect mRNA expression for cyclin D1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). RESULTS: The MTT assay showed that the number of living cells in the LSC, MSC and HSC groups was significantly smaller than that in the DMSO-treated group (P < 0.05) at 12-48 h. The inhibitory rate (IR) of the LSC group was 41.15% +/- 3.86%, 59.24% +/- 5.34% and 69.93% +/- 7.81% at 12, 24 and 48 h, respectively. The IR of the MSC group was 42.82% +/- 4.94%, 62.68% +/- 7.58% and 71.79% +/- 8.12% at 12, 24 and 48 h, respectively. The IR of the HSC group was 37.50% +/- 3.21%, 40.34% +/- 2.98% and 61.99% +/- 4.88% at 12, 24 and 48 h, respectively. These results suggested that a moderate dosage had the most obvious inhibitory efficacy at 48 h. Compared to the DMSO group, the mitosis index of the LSC, MSC, HSC groups was greatly decreased (P < 0.05) at all time points. Any dose of SC-B suppressed mitosis within 12-48 h. Compared to the DMSO group, the percentage of cells in the G0/G1 phase of the MSC group was greatly increased, and that of the S + G2M phase was greatly decreased, while the percentage of cell inhibition (PCI) in the MSC group was greatly increased (P < 0.05). This suggested that SC-B could exclusively arrest cells in the G0/G1 phase. Cyclin D1 mRNA expression was lower in the MSC group than that in the DMSO group (P < 0.05). CONCLUSION: SC-B can inhibit the proliferation and aberrant mitosis of human gastric cancer SCG-7901 cells in vitro. This inhibitory effect may be due to the down-regulation of cyclin D1 mRNA expression, which causes cell cycle arrest of gastric cancer cells.