[Optimization of processing technology of bark of Ilicis Rotundae Cortex based on quality by design concept].

Based on the concept of quality by design(QbD), this study optimized the processing technology of Ilicis Rotundae Cortex. According to the processing method and ingredient requirements of Ilicis Rotundae Cortex in the Chinese Pharmacopoeia, the content of syringin and pedunculoside, alcohol extract, fragmentation rate, and moisture content were taken as the critical quality attributes(CQAs). The soaking time, moistening time, and drying time were taken as critical process parameters(CPPs) by single factor tests. The weight coefficients of CQAs were determined by the analytic hierarchy process(AHP)-entropy weighting method, and the comprehensive score was calculated. With the comprehensive score as the response value, Box-Behnken design was employed to establish a mathematical model between CPPs and CQAs, and the design space for the processing of Ilicis Rotundae Cortex was built and verified. The results of ANOVA showed that the mathematical model had the P value below 0.05, the lack of fit greater than 0.05, adjusted R~2=0.910 5, and predicted R~2=0.831 0, which indicated that the proposed model had statistical significance and good prediction performance. Considering the factors in production, the best processing conditions of Ilicis Rotundae Cortex were decoction pieces of about 1 cm soaking for 1 h, moistening for 4 h, and drying at 60-70 ℃ in a blast drier for 2 h. The optimized processing technology of Ilicis Rotundae Cortex was stable and feasible, which can provide a reference for the standardized preparation and stable quality of Ilicis Rotundae Cortex.

Harnessing the Lysosomal Sorting Signals of the Cation-Independent Mannose-6-Phosphate Receptor for Targeted Degradation of Membrane Proteins.

Membrane proteins are a crucial class of therapeutic targets that remain challenging to modulate using traditional occupancy-driven inhibition strategies or current proteolysis-targeting degradation approaches. Here, we report that the inherent endolysosomal sorting machinery can be harnessed for the targeted degradation of membrane proteins. A new degradation technique, termed signal-mediated lysosome-targeting chimeras (SignalTACs), was developed by genetically fusing the signaling motif from the cation-independent mannose-6-phosphate receptor (CI-M6PR) to a membrane protein binder. Antibody-based SignalTACs were constructed with the CI-M6PR signal peptides fused to the C-terminus of both heavy and light chains of IgG. We demonstrated the scope of this platform technology by degrading five pathogenesis-related membrane proteins, including HER2, EGFR, PD-L1, CD20, and CD71. Furthermore, two simplified constructs of SignalTACs, nanobody-based and peptide-based SignalTACs, were created and shown to promote the lysosomal degradation of target membrane proteins. Compared to the parent antibodies, SignalTACs exhibited significantly higher efficiency in inhibiting tumor cell growth both in vitro and in vivo. This work provides a simple, general, and robust strategy for degrading membrane proteins with molecular precision and may represent a powerful platform with broad research and therapeutic applications.

Complete genome sequence of Bacillus Licheniformis NWMCC0046, a candidate for the laundry industry.

In this study, selected properties of protease and the complete genome sequence of Bacillus licheniformis NWMCC0046 were investigated, to discover laundry applications and other potential probiotic properties of this strain. Partial characterization of B. licheniformis NWMCC0046 showed that its protease has good activity both in alkaline environments and at low temperatures. Also, the protease is compatible with commercial detergents and can be used as a detergent additive for effective stain removal at low temperatures. The complete genome sequence of B. licheniformis NWMCC0046 is comprised of a 4,321,565 bp linear chromosome with a G + C content of 46.78% and no plasmids. It had 4504 protein-encoding genes, 81 transfer RNA (tRNA) genes, and 24 ribosomal RNA (rRNA) genes. Genomic analysis revealed genes involved in exocellular enzyme production and probiotic properties. In addition, genomic sequence analysis revealed specific genes encoding carbohydrate metabolism pathways, resistance, and cold adaptation capacity. Overall, protease properties show its potential as a detergent additive enzyme. The complete genome sequence information of B. licheniformis NWMCC0046 was obtained, and functional prediction revealed its numerous probiotic properties.

Functional modulation of an aquaporin to intensify photosynthesis and abrogate bacterial virulence in rice.

Plant aquaporins are a recently noted biological resource with a great potential to improve crop growth and defense traits. Here, we report the functional modulation of the rice (Oryza sativa) aquaporin OsPIP1;3 to enhance rice photosynthesis and grain production and to control bacterial blight and leaf streak, the most devastating worldwide bacterial diseases in the crop. We characterize OsPIP1;3 as a physiologically relevant CO2 -transporting facilitator, which supports 30% of rice photosynthesis on average. This role is nullified by interaction of OsPIP1;3 with the bacterial protein Hpa1, an essential component of the Type III translocon that supports translocation of the bacterial Type III effectors PthXo1 and TALi into rice cells to induce leaf blight and streak, respectively. Hpa1 binding shifts OsPIP1;3 from CO2 transport to effector translocation, aggravates bacterial virulence, and blocks rice photosynthesis. On the contrary, the external application of isolated Hpa1 to rice plants effectively prevents OsPIP1;3 from interaction with Hpa1 secreted by the bacteria that are infecting the plants. Blockage of the OsPIP1;3-Hpa1 interaction reverts OsPIP1;3 from effector translocation to CO2 transport, abrogates bacterial virulence, and meanwhile induces defense responses in rice. These beneficial effects can combine to enhance photosynthesis by 29-30%, reduce bacterial disease by 58-75%, and increase grain yield by 11-34% in different rice varieties investigated in small-scale field trials conducted during the past years. Our results suggest that crop productivity and immunity can be coordinated by modulating the physiological and pathological functions of a single aquaporin to break the growth-defense tradeoff barrier.

Indole-Based Long-Wavelength Fluorescent Probes for Bioimaging of S-Nitrosylation in Mitochondria.

S-Nitrosylation has been found to play an important role in regulating mitochondrial function. However, probes for detection of protein S-nitrosylation in mitochondria remain unexplored. Herein, a novel 4-(pyridin-4-yl)vinyl-substituted indole was designed, exhibiting a long-wavelength emission and a high fluorescent quantum yield. Functionalization of the 7-position of the indole ring with an arylphosphine ester resulted with probes with efficient mitochondria-targeting ability. Furthermore, the indole-arylphosphine displayed a significant fluorescence enhancement upon exposure to S-nitrosoglutathione (GSNO) at low micromolar concentrations in A431 cells. Taken together, this study provides a new indole-based fluorescent probe with a unique long-wavelength emission for direct detection of S-nitrosylation in mitochondria, which may represent a powerful tool for understanding the critical roles of S-nitrosylation within mitochondria of living organisms.

Peroxisome Proliferator FpPEX11 Is Involved in the Development and Pathogenicity in Fusarium pseudograminearum.

Fusarium crown rot (FCR) of wheat, an important soil-borne disease, presents a worsening trend year by year, posing a significant threat to wheat production. Fusarium pseudograminearum cv. b was reported to be the dominant pathogen of FCR in China. Peroxisomes are single-membrane organelles in eukaryotes that are involved in many important biochemical metabolic processes, including fatty acid ß-oxidation. PEX11 is important proteins in peroxisome proliferation, while less is known in the fungus F. pseudograminearum. The functions of FpPEX11a, FpPEX11b, and FpPEX11c in F. pseudograminearum were studied using reverse genetics, and the results showed that FpPEX11a and FpPEX11b are involved in the regulation of vegetative growth and asexual reproduction. After deleting FpPEX11a and FpPEX11b, cell wall integrity was impaired, cellular metabolism processes including active oxygen metabolism and fatty acid ß-oxidation were significantly blocked, and the production ability of deoxynivalenol (DON) decreased. In addition, the deletion of genes of FpPEX11a and FpPEX11b revealed a strongly decreased expression level of peroxisome-proliferation-associated genes and DON-synthesis-related genes. However, deletion of FpPEX11c did not significantly affect these metabolic processes. Deletion of the three protein-coding genes resulted in reduced pathogenicity of F. pseudograminearum. In summary, FpPEX11a and FpPEX11b play crucial roles in the growth and development, asexual reproduction, pathogenicity, active oxygen accumulation, and fatty acid utilization in F. pseudograminearum.

FgPsd2, a phosphatidylserine decarboxylase of Fusarium graminearum, regulates development and virulence.

Phosphatidylserine decarboxylases (Psds) are enzymes regulating phosphatidylethanolamine biosynthesis in prokaryotes and eukaryotes, and have the central role in lipid metabolism. To date, the functions of Psds in plant pathogenic fungi are not fully understood. In this study, we have characterized two yeast Psd orthologues: FgPsd1 and FgPsd2, in Fusarium graminearum. Our results indicate that FgPsd1 and FgPsd2 are localized in mitochondria and Golgi, respectively. In addition, we have determined that FgPsd1 is a lethal gene and deletion of FgPsd2 resulted in a significant reduction of mycelial growth and conidiation. Futhermore, the FgPsd2 deletion mutant (ΔFgPsd2) is defective in ascospore production and virulence in wheat. Our study has also found that the ΔFgPsd2 mutant is more sensitive to osmotic and oxygen stresses. Moreover, deletion of FgPsd2 reduced the formation of lipid droplets and aggravated autophagy in F. graminearum. In summary, our findings indicate that FgPsd2 is important for mycelial growth, sexual and asexual reproduction, virulence, lipid droplet formation and autophagy in F. graminearum.

Correction: Pan et al. Genome-Wide Identification of M35 Family Metalloproteases in Rhizoctonia cerealis and Functional Analysis of RcMEP2 as a Virulence Factor during the Fungal Infection to Wheat. Int. J. Mol. Sci. 2020, 21, 2984.

In the original article, there was a mistake in Figure 8 as published [...].

Gastroprotective effect of cirsilineol against hydrochloric acid/ethanol-induced gastric ulcer in rats.

This study was designed to evaluate the gastroprotective activity of cirsilineol in hydrochloric acid (HCl)/ethanol-induced gastric ulcer model. Cirsilineol was administered at the doses of 20 and 40 mg/kg in HCl/ethanol-induced rats. The gastroprotective ability was verified by determining the ulcer score, total acidity, hemoglobin, inflammatory cytokines, lipid peroxides, and enzymatic antioxidants superoxide dismutase (SOD) and catalase (CAT) in gastric tissue and serum biochemical analysis. The results showed a favorable increase in the hemoglobin level, antioxidant enzymes (SOD and CAT), restored electrochemical balance (carbon dioxide & anion gap) while a noticeable decrease in ulcer index, total acidity, lipid peroxides, inflammatory cytokines (interleukin-1 beta [IL-1ß], IL-6, and tumor necrosis factor alpha) in rats treated with the cirsilineol. The serum biochemical analysis on liver markers (alkaline phosphatases, alanine aminotransferase, and aspartate aminotransferase), kidney markers (urea, creatinine, albumin, globulin, total protein), and lipid profile (triglyceride, high-density lipoprotein, total cholesterol) were attenuated by cirsilineol treatment in rats. Histopathology showed enhanced gastric protection and preserved the integrity of gastric mucosa upon cirsilineol administration. These results ultimately suggest that cirsilineol has gastroprotective effects that prevent the development of gastric ulcer.

Mitochondrial FgEch1 is responsible for conidiation and full virulence in Fusarium graminearum.

Enoyl-CoA hydratase (Ech) is an important and well-recognized enzyme that functions in the degradation of fatty acids by ß-oxidation. However, its functions in plant pathogenic fungi are not well known. We characterized an Ech1 orthologue, FgEch1, in Fusarium graminearum. The FgEch1 deletion mutant was defective in the utilization of short-chain fatty acids and conidiation, but not in hyphal growth on glucose-rich media or in perithecium formation. The FgEch1 deletion mutant showed reduced deoxynivalenol (DON) production and virulence in plants. Deletion of FgEch1 also led to increased production of lipid droplets and autophagy. FgEch1, which was localized in the mitochondrion, required the MTS domain for mitochondrial localization and function in F. graminearum. Taken together, these data indicate that mitochondrial FgEch1 is important for conidiation, DON production, and plant infection.

The roles of FgPEX2 and FgPEX12 in virulence and lipid metabolism in Fusarium graminearum.

Fusarium head blight (FHB) is a wheat disease with a worldwide prevalence, caused by Fusarium graminearum. Peroxisomes are ubiquitous in eukaryotic cells and are involved in various biochemical phenomena. FgPEX2 and FgPEX12 encode RING-finger peroxins PEX2 and PEX12 in F. graminearum. This study aimed to functionally characterize FgPEX2 and FgPEX12 in F. graminearum. We constructed deletion mutants of FgPEX2 and FgPEX12 via homologous recombination. The ΔPEX2 and ΔPEX12 mutants displayed defects in sexual and asexual development, virulence, cell wall integrity (CWI), and lipid metabolism. Deletion of FgPEX2 and FgPEX12 significantly decreased deoxynivalenol production. Furthermore, fluorescence microscopic analysis of the subcellular localization of GFP-PMP70 and GFP-HEX1 revealed that FgPEX2 and FgPEX12 maintain Woronin bodies. These results show that FgPEX2 and FgPEX12 are required for growth, conidiation, virulence, cell wall integrity, and lipid metabolism in F. graminearum and do not influence their peroxisomes.

Endocytic FgEde1 regulates virulence and autophagy in Fusarium graminearum.

Endocytosis plays critical roles in cellular processes, including nutrient uptake and signal transduction. Ede1 is an endocytic scaffolding protein that contributes to endocytic site initiation and maturation in yeast. However, the functions of Ede1 in phytopathogenic fungi are not known. Here, we identified functions of FgEde1 (FGSG_05182) in Fusarium graminearum. Deletion of FgEde1 resulted in defects in hyphal growth, conidiation and ascospore development. The FgEde1 deletion mutant showed reduced deoxynivalenol (DON) production and virulence in wheat. Furthermore, the FgEde1 deletion mutant also exhibited increased resistance to osmotic and oxidative stress as well as cell-wall perturbing agents. Importantly, deletion of FgEde1 increased the severity of autophagy in hyphae. Taken together, these results reveal that FgEde1 is involved in hyphal growth, asexual and sexual reproduction, virulence, stress responses, and autophagy in F. graminearum.

Generation of mice with hepatocyte-specific conditional deletion of sphingosine kinase 1.

SphK1 gene has different roles in various types of cells in liver diseases, but most studies are based on global knockout mice, which hampers the study on the cellular and molecular mechanisms of SphK1. In order to further study the role of SphK1 in liver, SphK1 conditional knockout mice were constructed. A liver-specific SphK1 gene knockout mouse model was constructed by the Cre/Loxp recombinant enzyme system. PCR technologies and western blotting were used to identified the elimination of SphK1 gene in hepatocytes. SphK1flox/flox mice were used as a control group to verify the effectiveness of SphK1 liver-specific knockout mice from the profile, pathology, and serology of mice. The ablation of SphK1 in hepatic parenchymal cells was demonstrated by fluorescent in situ hybridization and the contents of S1P and Sph were measured by ELISA kit. The genotypes of liver in SphK1 conditional knockout mice were different from that of other organs. The mRNA and protein levels of SphK1 in liver tissue of SphK1 conditional knockout mice were almost depleted by compared with SphK1flox/flox mice. Physiology and pathology showed no significant difference between SphK1 liver conditional knockout mice and SphK1flox/flox mice. Additionally, SphK1 was eliminated in hepatocytes, leading to the reduce of S1P content in hepatocytes and liver tissues and the increase of Sph content in hepatocytes. The model of SphK1 gene liver conditional knockout mice was successfully constructed, providing a tool for the study of the roles of SphK1 in hepatocyte and liver diseases.

Extracellular Vesicle-Transported Long Non-Coding RNA (LncRNA) X Inactive-Specific Transcript (XIST) in Serum is a Potential Novel Biomarker for Colorectal Cancer Diagnosis.

BACKGROUND Colorectal cancer (CRC) cell-derived extracellular vesicles (EVs) contribute to tumor progression. Differentially expressed long non-coding (lnc)RNAs may serve as biomarkers for CRC diagnosis. This study aimed to discuss the diagnostic value of serum EV-derived lncRNA X inactive-specific transcript (XIST) in CRC. MATERIAL AND METHODS Serum EVs were extracted and identified. Microarray analysis was performed to screen out the differentially expressed lncRNAs in serum EVs. The expression and diagnostic efficacy of the most differentially expressed lncRNA were measured. Kaplan-Meier survival analysis was performed to evaluate the association between survival time and XIST expression in EVs. The expression profile of serum EV-carried XIST in 94 CRC patients with different tumor-node-metastasis stages, lymph node metastasis, and differentiation was assessed. The serum contents of CEA, CA242, CA199, and CA153 were measured. RESULTS XIST in serum EVs in CRC patients was upregulated, with greatest diagnostic value. CRC patients with higher expression of XIST in serum EVs had worse 5-year survival rates and shorter life cycles, lower differentiation, higher lymph node metastasis, and tumor-node-metastasis than patients with lower XIST expression. XIST expression in serum EVs was positively correlated with CRC marker contents. CONCLUSIONS XIST upregulation in serum EVs is related to CRC progression, which may be helpful to the clinical diagnosis and prognosis of CRC.

The Cysteine-Rich Repeat Protein TaCRR1 Participates in Defense against Both Rhizoctonia cerealis and Bipolaris sorokiniana in Wheat.

The domain of unknown function 26 (DUF26), harboring a conserved cysteine-rich motif (C-X8-C-X2-C), is unique to land plants. Several cysteine-rich repeat proteins (CRRs), belonging to DUF26-containing proteins, have been implicated in the defense against fungal pathogens in ginkgo, cotton, and maize. However, little is known about the functional roles of CRRs in the important staple crop wheat (Triticum aestivum). In this study, we identified a wheat CRR-encoding gene TaCRR1 through transcriptomic analysis, and dissected the defense role of TaCRR1 against the soil-borne fungi Rhizoctonia cerealis and Bipolaris sorokiniana, causal pathogens of destructive wheat diseases. TaCRR1 transcription was up-regulated in wheat towards B. Sorokiniana or R. cerealis infection. The deduced TaCRR1 protein contained a signal peptide and two DUF26 domains. Heterologously-expressed TaCRR1 protein markedly inhibited the mycelia growth of B. sorokiniana and R. cerealis. Furthermore, the silencing of TaCRR1 both impaired host resistance to B. sorokiniana and R. cerealis and repressed the expression of several pathogenesis-related genes in wheat. These results suggest that the TaCRR1 positively participated in wheat defense against both B. sorokiniana and R. cerealis through its antifungal activity and modulating expression of pathogenesis-related genes. Thus, TaCRR1 is a candidate gene for improving wheat resistance to B. sorokiniana and R. cerealis.

Genome-Wide Identification of M35 Family Metalloproteases in Rhizoctonia cerealis and Functional Analysis of RcMEP2 as a Virulence Factor during the Fungal Infection to Wheat.

Rhizoctonia cerealis is the causal pathogen of the devastating disease, sharp eyespot, of the important crop wheat (Triticum aestivum L.). In phytopathogenic fungi, several M36 metalloproteases have been implicated in virulence, but pathogenesis roles of M35 family metalloproteases are largely unknown. Here, we identified four M35 family metalloproteases from R. cerealis genome, designated RcMEP2-RcMEP5, measured their transcriptional profiles, and investigated RcMEP2 function. RcMEP2-RcMEP5 are predicted as secreted metalloproteases since each protein sequence contains a signal peptide and an M35 domain that includes two characteristic motifs HEXXE and GTXDXXYG. Transcription levels of RcMEP2-RcMEP5 markedly elevated during the fungus infection to wheat, among which RcMEP2 expressed with the highest level. Functional dissection indicated that RcMEP2 and its M35 domain could trigger H2O2 rapidly-excessive accumulation, induce cell death, and inhibit expression of host chitinases. This consequently enhanced the susceptibility of wheat to R. cerealis and the predicated signal peptide of RcMEP2 functions required for secretion and cell death-induction. These results demonstrate that RcMEP2 is a virulence factor and that its M35 domain and signal peptide are necessary for the virulence role of RcMEP2. This study facilitates a better understanding of the pathogenesis mechanism of metalloproteases in phytopathogens including R. cerealis.

FgPEX4 is involved in development, pathogenicity, and cell wall integrity in Fusarium graminearum.

Peroxisomes are indispensable organelles that play critical roles in various biological processes in eukaryotic cells. PEX4, one of the peroxins, is the ubiquitin-conjugating enzyme. To functionally characterize roles of FgPEX4 in the phytopathogenic fungus, Fusarium graminearum, we constructed a deletion mutant of FgPEX4 (ΔPEX4) through homologous recombination. ΔPEX4 displayed reduced mycelial growth, conidiation, and the production of perithecia. ΔPEX4 was defective in pathogenicity and production of the mycotoxin deoxynivalenol (DON). In addition, FgPEX4 was involved in cell wall integrity, lipid droplet accumulation, and the elimination of reactive oxygen species. Western blot analysis revealed reduced phosphorylation of Mgv1 in the ∆PEX4 mutant. Importantly, proteomics analysis indicated that protein expression levels related to protein biosynthesis, fatty acid metabolism, cell wall synthesis, and oxidation-reduction reactions were downregulated in ΔPEX4 compared with the wild type. Taken together, these results demonstrate that FgPEX4 is important for development, pathogenicity, and cell wall integrity.

FgPEX1 and FgPEX10 are required for the maintenance of Woronin bodies and full virulence of Fusarium graminearum.

Peroxisomes are ubiquitous single-membrane-bound organelles that perform a variety of biochemical functions in eukaryotic cells. Proteins involved in peroxisomal biogenesis are collectively called peroxins. Currently, functions of most peroxins in phytopathogenic fungi are poorly understood. Here, we report identification of PEX1 and PEX10 in the phytopathogenic fungus, Fusarium graminearum, namely FgPEX1 and FgPEX10, the orthologs of yeast ScPEX1 and ScPEX10. To functionally characterize FgPEX1 and FgPEX10, we constructed deletion mutants of FgPEX1 and FgPEX10 (ΔPEX1 and ΔPEX10) by targeting gene-replacement strategies. Our data demonstrate that both mutants displayed reduced mycelial growth, conidiation, and production of perithecia. Deletion of FgPEX1 and FgPEX10 resulted in a shortage of acetyl-CoA, which is an important reason for the reduced deoxynivalenol production and inhibited virulence of F. graminearum. Moreover, ΔPEX1 and ΔPEX10 showed an increased accumulation of lipid droplets and endogenous reactive oxygen species. In addition, FgPEX1 and FgPEX10 were found to be involved in the maintenance of cell wall integrity and Woronin bodies.

AidB, a Novel Thermostable N-Acylhomoserine Lactonase from the Bacterium Bosea sp.

Many Gram-negative bacteria employ N-acylhomoserine lactones (AHLs) as quorum-sensing (QS) signal molecules to regulate virulence expression in a density-dependent manner. Quorum quenching (QQ) via enzymatic inactivation of AHLs is a promising strategy to reduce bacterial infections and drug resistance. Herein, a thermostable AHL lactonase (AidB), which could degrade different AHLs, with or without a substitution of carbonyl or hydroxyl at the C-3 position, was identified from the soil bacterium Bosea sp. strain F3-2. Ultrahigh-performance liquid chromatography analysis demonstrated that AidB is an AHL lactonase that hydrolyzes the ester bond of the homoserine lactone (HSL) ring. AidB was thermostable in the range 30 to 80°C and showed maximum activity after preincubation at 60°C for 30 min. The optimum temperature of AidB was 60°C, and the enzyme could be stably stored in double-distilled water (ddH2O) at 4°C or room temperature. AidB homologs were found only in Rhizobiales and Rhodospirillales of the Alphaproteobacteria AidB from Agrobacterium tumefaciens and AidB from Rhizobium multihospitium (with amino acid identities of 50.6% and 52.8% to AidB, respectively) also showed thermostable AHL degradation activity. When introduced into bacteria, plasmid-expressed AidB attenuated pyocyanin production by Pseudomonas aeruginosa PAO1 and the pathogenicity of Pectobacterium carotovorum subsp. carotovorum Z3-3, suggesting that AidB is a potential therapeutic agent by degrading AHLs.IMPORTANCE A quorum-sensing system using AHLs as the signal in many bacterial pathogens is a critical virulence regulator and an attractive target for anti-infective drugs. In this work, we identified a novel AHL lactonase, AidB, from a soil bacterial strain, Bosea sp. F3-2. The expression of aidB reduced the production of AHL signals and QS-dependent virulence factors in Pseudomonas aeruginosa and Pectobacterium carotovorum The homologs of AidB with AHL-degrading activities were found only in several genera belonging to the Alphaproteobacteria Remarkably, AidB is a thermostable enzyme that retained its catalytic activity after treatment at 80°C for 30 min and exhibits reliable storage stability at both 4°C and room temperature. These properties might make it more suitable for practical application.

The Eno Gene of Burkholderia cenocepacia Strain 71-2 is Involved in Phosphate Solubilization.

Bacterial strain 71-2 with phosphate-solubilizing activity was isolated from tobacco rhizosphere and classified as Burkholderia cenocepacia based on sequence analyses of 16S rRNA and recA genes. To learn phosphate-solubilizing mechanisms of 71-2, mutants showing reduced solubilizing phosphate activity were obtained using the EZ-Tn5 transposon. Mutant 71-2-MT51 was reduced in the solubilizing phosphate content to 34.36% as compared with the wild-type strain 71-2. The disrupted gene in 71-2-MT51 was cloned and sequenced, and the putative protein from the gene shared 65.26% identity to protein sequences of enolase from Escherichia coli, which suggests the gene encodes an enzyme of enolase. Complementation analyzing showed that Eno was responsible for phosphate solubilizing for B. cenocepacia strain 71-2. To our knowledge, this is the first report of Eno involved in phosphate solubilizing in B. cenocepacia as well as in other bacteria.