A pyocin-like T6SS effector mediates bacterial competition in Yersinia pseudotuberculosis.

Within the realm of Gram-negative bacteria, bacteriocins are secreted almost everywhere, and the most representative are colicin and pyocin, which are secreted by Escherichia coli and Pseudomonas aeruginosa, respectively. Signal peptides at the amino terminus of bacteriocins or ABC transporters can secrete bacteriocins, which then enter bacteria through cell membrane receptors and exert toxicity. In general, the bactericidal spectrum is usually narrow, killing only the kin or closely related species. Our previous research indicates that YPK_0952 is an effector of the third Type VI secretion system (T6SS-3) in Yersinia pseudotuberculosis. Next, we sought to determine its identity and characterize its toxicity. We found that YPK_0952 (a pyocin-like effector) can achieve intra-species and inter-species competitive advantages through both contact-dependent and contact-independent mechanisms mediated by the T6SS-3 while enhancing the intestinal colonization capacity of Y. pseudotuberculosis. We further identified YPK_0952 as a DNase dependent on Mg2+, Ni2+, Mn2+, and Co2+ bivalent metal ions, and the homologous immune protein YPK_0953 can inhibit its activity. In summary, YPK_0952 exerts toxicity by degrading nucleic acids from competing cells, and YPK_0953 prevents self-attack in Y. pseudotuberculosis.IMPORTANCEBacteriocins secreted by Gram-negative bacteria generally enter cells through specific interactions on the cell surface, resulting in a narrow bactericidal spectrum. First, we identified a new pyocin-like effector protein, YPK_0952, in the third Type VI secretion system (T6SS-3) of Yersinia pseudotuberculosis. YPK_0952 is secreted by T6SS-3 and can exert DNase activity through contact-dependent and contact-independent entry into nearby cells of the same and other species (e.g., Escherichia coli) to help Y. pseudotuberculosis to exert a competitive advantage and promote intestinal colonization. This discovery lays the foundation for an in-depth study of the different effector protein types within the T6SS and their complexity in competing interactions. At the same time, this study provides a new development for the toolbox of toxin/immune pairs for studying Gram-negative bacteriocin translocation.

Trojan horselike T6SS effector TepC mediates both interference competition and exploitative competition.

The type VI secretion system (T6SS) is a bacterial weapon capable of delivering antibacterial effectors to kill competing cells for interference competition, as well as secreting metal ion scavenging effectors to acquire essential micronutrients for exploitation competition. However, no T6SS effectors that can mediate both interference competition and exploitation competition have been reported. In this study, we identified a unique T6SS-1 effector in Yersinia pseudotuberculosis named TepC, which plays versatile roles in microbial communities. First, secreted TepC acts as a proteinaceous siderophore that binds to iron and mediates exploitative competition. Additionally, we discovered that TepC has DNase activity, which gives it both contact-dependent and contact-independent interference competition abilities. In conditions where iron is limited, the iron-loaded TepC is taken up by target cells expressing the outer membrane receptor TdsR. For kin cells encoding the cognate immunity protein TipC, TepC facilitates iron acquisition, and its toxic effects are neutralized. On the other hand, nonkin cells lacking TipC are enticed to uptake TepC and are killed by its DNase activity. Therefore, we have uncovered a T6SS effector, TepC, that functions like a "Trojan horse" by binding to iron ions to provide a valuable resource to kin cells, whereas punishing cheaters that do not produce public goods. This lure-to-kill mechanism, mediated by a bifunctional T6SS effector, may offer new insights into the molecular mechanisms that maintain stability in microbial communities.

Determining nitrogen topdressing rates in accordance with actual seedling density and crop nitrogen status in direct-seeded rice.

BACKGROUND: Adjusting nitrogen (N) input based on actual seedling density (ASD) and plant N status is a practical approach for improving the yield stability of direct-seeded rice. However, the adjustment of topdressing N rates has been empirical in the past. This study aimed to establish a quantitative approach for determining N topdressing rates during tillering (Ntil ) and panicle development (NPI ) based on ASD and crop N status in direct-seeded rice. Field experiments were conducted involving 12 treatments, consisting of four Ntil and three seeding rates in 2017, and eight treatments combining seeding rate, Ntil , and NPI in 2020. RESULTS: Linear regression analysis revealed that the tiller number at panicle initiation (TILPI ) was predominantly influenced by ASD and Ntil . The determination coefficients (R2 ) of the regression models ranged from 0.887 to 0.936 across the four-season experiments. The results indicated that Ntil could be determined accurately using ASD and the target maximum tiller number. Similarly, grain yield was influenced significantly by the N uptake at panicle initiation (NUPPI ) and NPI , with R2 of 0.814 and 0.783 in the early and late seasons of 2020, respectively. This suggested that NPI could be calculated based on NUPPI and the target grain yield. CONCLUSION: The findings offer a quantitative method for establishing N topdressing rates for tillering and panicle development, relying on the monitoring of actual seedling density and plant N status in direct-seeded rice production. © 2023 Society of Chemical Industry.

Improving grain yield and nitrogen use efficiency of direct-seeded rice with simplified and nitrogen-reduced practices under a double-cropping system in South China.

BACKGROUND: Enhancing grain yield and nitrogen use efficiency (NUE) of rice is of great importance for sustainable agricultural development. Little effort has been made to increase grain yield and NUE of direct-seeded rice under the double-cropping system in South China. Field trials were conducted during 2018-2020 with four treatments, including nitrogen-free, farmers' fertilization practice (FP), 'three controls' nutrient management (TC), and simplified and nitrogen-reduced practice (SNRP). RESULTS: Grain yield under SNRP averaged 6.46 t ha-1 during the three years and was 23.0% higher than that of FP but comparable to that of TC. Recovery efficiency (REN ), agronomic efficiency (AEN ), and partial factor productivity (PFPN ) of nitrogen under SNRP increased by 12.0-22.7%, 159.3-295.0% and 94.6-112.5% respectively compared with FP. Harvest index and sink capacity increased by 7.3-10.8% and 14.9-21.3% respectively. Percentage of productive tillers (PPT) and biomass after heading increased by 24.0% and 104.5% respectively. Leaf nitrogen concentration at heading and nitrogen accumulation after heading increased by 16.3% and 842.0% respectively. Grain yield was positively correlated with PPT, sink capacity, harvest index, biomass and nitrogen accumulation after heading, REN , AEN , and PFPN . CONCLUSION: Grain yield and NUE under SNRP were superior to those under FP and comparable to those under TC. Increase in sink capacity, higher PPT, more biomass and nitrogen accumulation after heading, and greater harvest index were responsible for high grain yield and NUE in SNRP with reduced nitrogen fertilizer and labor input. SNRP is a feasible approach for direct-seeded rice under a double-cropping system in South China. © 2023 Society of Chemical Industry.

Growth, nutrient uptake and transcriptome profiling of rice seedlings in response to mixed provision of ammonium- and nitrate-nitrogen.

Nitrogen (N) is a principal macronutrient and plays a paramount role in mineral nutrition of rice plants. Mixed provision of ammonium- and nitrate-nitrogen (MPAN) at a moderate level could enhance N uptake and translocation and promote growth of rice, but current understanding of their molecular mechanisms is still insufficient. Two rice lines of W6827 and GH751, with contrasting ability of N uptake, were subjected to four levels of MPAN (NH4+/NO3- = 100:0, 75:25, 50:50, 25:75) in hydroponic experiments. In terms of plant height, growth rate and shoot biomass, growth of GH751 tended to increase firstly and then decrease with enhancement in NO3--N ratio. It attained maximal level under 75:25 MPAN, with an 8.3% increase in shoot biomass. In general, W6827 was comparatively less responsive to MPAN. For GH751, the uptake rate of N, phosphor (P) and potassium (K) under 75:25 MPAN was enhanced by 21.1%, 20.8% and 16.1% in comparison with that of control (100:0 MPAN). Meanwhile, the translocation coefficient and content in shoots of N, P and K were all increased significantly. In contrast to transcriptomic profile under control, 288 differentially expressed genes (DEGs) were detected to be up-regulated and 179 DEGs down-regulated in transcription under 75:25 MPAN. Gene Ontology analysis revealed that some DEGs were up-regulated under 75:25 MPAN and they code for proteins mainly located in membrane and integral component of membrane and involved in metal ion binding, oxidoreductase activity and other biological processes. KEGG pathway enrichment analysis indicated that DEGs related to nitrogen metabolism, carbon fixation in photosynthetic organisms, photosynthesis, starch and sucrose metabolism, and zeatin biosynthesis were up- or down-regulated in transcription under 75:25 MPAN, and they are responsible for improved nutrient uptake and translocation and enhanced growth of seedlings.

Allometric relationships between leaf and petiole traits across 31 floating-leaved plants reveal a different adaptation pattern from terrestrial plants.

BACKGROUND AND AIMS: Allometric scaling between stomata and xylem for terrestrial woody plants is a widely observed pattern that may be constrained by water transport. Floating-leaved plants, a particular life form of aquatic plants, have leaves in direct contact with both air and water and a poorly developed xylem that may not be limited by water supply as for terrestrial plants. However, whether such an allometric scaling relationship still exists in floating-leaved plants has not been explored. METHODS: We analysed 31 floating-leaved species/varieties with a range in leaf area covering six orders of magnitude. For all 31 floating-leaved plants, we studied the allometric relationships between leaf area and petiole transverse area, and between total stomatal area and petiole vascular area. KEY RESULTS: The slopes of both relationships were similar to the slope of the allometric relationship (1.23) between total stomatal area and xylem area of 53 terrestrial plants. However, for ten of them with xylem that can be clearly defined, the strong positive relationship between total stomatal area and petiole xylem area had a significantly smaller slope than that of terrestrial plants (0.64 vs. 1.23). Furthermore, after considering phylogeny, the scaling relationships between total stomatal area and petiole traits in floating-leaved plants remained significant. CONCLUSIONS: We speculated that for floating-leaved plants, the hyperallometric relationship (slope >1) between the construction of leaf/stoma and petiole was promoted by the high demand for photosynthesis and thus more leaves/stomata. While the hypoallometric relationship (slope <1) between stomatal and xylem area was related more to hydraulic processes, the selection pressure on stomata was lower than xylem of floating-leaved plants. Allometric relationships among the hydraulic traits on water transport of aquatic plants are the result of natural selection to achieve maximum carbon gain, which is similar to terrestrial plants.

Phloem Unloading in Developing Rice Caryopses and its Contribution to Non-Structural Carbohydrate Translocation from Stems and Grain Yield Formation.

Phloem unloading plays an important role in photoassimilate partitioning and grain yield improvements in cereal crops. The phloem unloading strategy and its effects on photoassimilate translocation and yield formation remain unclear in rice. In this study, plasmodesmata were observed at the interface between the sieve elements (SEs) and companion cells (CCs), and between the SE-CC complex and surrounding parenchyma cells (PCs) in phloem of the dorsal vascular bundle in developing caryopses. Carboxyfluorescein (CF) signal was detected in the phloem of caryopses, which showed that CF was unloaded into caryopses. These results indicated that the SE-CC complex was symplasmically connected with adjacent PCs by plasmodesmata. Gene expression for sucrose transporter (SUT) and cell wall invertase (CWI), and OsSUT1 and OsCIN1 proteins were detected in developing caryopses, indicating that rice plants might actively unload sucrose into caryopses by the apoplasmic pathway. Among three rice recombinant inbred lines, R201 exhibited lower plasmodesmal densities at the boundaries between cell types (SE-CC, SE-PC and CC-PC) in developing caryopses than R91 and R156. R201 also had lower expression of SUT and CWI genes and lower protein levels of OsSUT1 and OsCIN1, as well as CWI activity, than R91 and R156. These data agreed with stem non-structural carbohydrate (NSC) translocation and grain yields for the three lines. The nitrogen application rate had no significant effect on plasmodesmal densities at the interfaces between different cells types, and did not affect CF unloading in the phloem of developing caryopses. Low nitrogen treatment enhanced expression levels of OsSUT and OsCIN genes in the three lines. These results suggested that nitrogen application had no substantial effect on symplasmic unloading but affected apoplasmic unloading. Therefore, we concluded that poor symplasmic and apoplasmic unloading in developing caryopses might result in low stem NSC translocation and poor grain yield formation of R201.

Impacts of Chronic Habitat Fragmentation on Genetic Diversity of Natural Populations of Prunus persica in China.

Wild peach is an important resource for improving existing peach varieties. However, the extant populations of wild peach show fragmented distribution due to human disturbance and geographic isolation. In this study, we used natural populations (or wild populations) of Prunus persica (Rosaceae) to assess the genetic effects of habitat fragmentation. A total of 368 individuals sampled from 16 natural populations were analyzed using 23 polymorphic simple sequence repeat (SSR) markers. Prunus persica maintained low within-population genetic variation and high level of genetic differentiation. Two genetic clusters were revealed based on three different methods (UPGMA, PCoA, and STRUCTURE). All populations showed a significant heterozygosity deficiency and most extant populations experienced recent reduction in population size. A significant isolation by distance (IBD) was observed with Mantel's test. Compared to historical gene flow, contemporary gene flow was restricted among the studied populations, suggesting a decrease in gene flow due to habitat fragmentation. Habitat fragmentation has impacted population genetic variation and genetic structure of P. persica. For breeding and conservation purpose, collecting as many individuals as possible from multiple populations to maximize genetic diversity was recommended during the process of germplasm collection. In addition, populations from central China had higher genetic diversity, suggesting these populations should be given priority for conservation and germplasm collection.

Stem small vascular bundles have greater accumulation and translocation of non-structural carbohydrates than large vascular bundles in rice.

Phloem unloading and loading are associated with stem non-structural carbohydrates (NSCs) accumulation and remobilization in rice (Oryza sativa L.). Four rice recombinant inbred lines (R032, R191, R046, and R146) derived from a cross between Zhenshan 97 and Minghui 63 were used to investigate the contributions of stem large and small vascular bundles (SVBs) to NSCs accumulation and translocation. Before heading, the parenchyma cells in stem cortex tissues (PCs) surrounding SVBs had higher starch density than those surrounding large vascular bundles (LVBs). Moreover, the protein levels of sucrose transporters (SUTs), cell wall invertase, sucrose synthase, and adenosine diphosphate glucose pyrophosphorylase, as well as the phloem plasmodesma densities were higher in SVBs than those in LVBs. After heading, starch density decreased more in PCs surrounding SVBs than in LVBs. Also, the protein levels of SUTs, α-amylase, sucrose phosphate synthase and sucrose synthase, the phloem plasmodesma densities in SVBs were higher than those in LVBs. The correlations of the number and total cross-sectional area of SVBs with mass and contribution to yield of transferred NSCs were higher than those of LVBs. Our results suggest that SVBs may have higher contributions to pre-anthesis stem NSCs accumulation and post-anthesis translocation than LVBs, which is potentially attributed to the high level of protein and enzyme involved in stem unloading and loading via apoplastic and symplastic pathways.

K fertilizer alleviates N2O emissions by regulating the abundance of nitrifying and denitrifying microbial communities in the soil-plant system.

Potassium (K) fertilizer additions can result in high crop yields of good quality and low nitrogen (N) loss; however, the interaction between K and N fertilizer and its effect on N2O emissions and associated microbes remain unclear. We investigated this in a pot experiment with six fertilizer treatments involving K and two sources of N, using agricultural soil from the suburbs of Wuhan, central China. The aim was to determine the effects of the interaction between K and different forms of N on the N2O flux and the abundance of nitrifying and denitrifying microbial communities, using static chamber-gas chromatography and high-throughput sequencing methods. Compared with no fertilizer control (CK), the addition of nitrate fertilizer (NN) or ammonia fertilizer (AN) or K fertilizer significantly increased N2O emissions. However, the combined application (NNK) of K and NN significantly reduced the average N2O emissions by 28.3%, while the combined application (ANK) of K and AN increased N2O emissions by 22.7%. The abundance of nitrifying genes amoA in ammonia oxidizing archaea (AOA) and ammonia oxidizing bacteria (AOB) changed in response to N and/or K fertilization, but the denitrifying genes narG, nirK and norl were strongly correlated with N2O emissions. This suggests that N or K fertilizer and their interaction affect N2O emissions mainly by altering the abundance of functional genes of denitrifying microbes in the soil-plant system. The genera Paracoccus, Rubrivivax and Geobacter as well as Streptomyces and Hyphomicrobium play an important role in N2O emissions during denitrification with the combined application of N and K.

The transcriptional regulator Zur regulates the expression of ZnuABC and T6SS4 in response to stresses in Yersinia pseudotuberculosis.

Zinc homeostasis is crucial for the development and stress resistance of bacteria in the environment. Serial zinc sensing transcriptional regulators, zinc transporters and zinc binding proteins were found to maintain the zinc homeostasis in bacteria. Zur is a zinc uptake regulator that is widely distributed in species, and ZnuABC, as well as the Type VI Secretion System (T6SS4) function in zinc acquisition. Here, we report that the regulator Zur inhibits the expression of the ZnuABC which inhibition could be eliminated at low zinc level, and upregulates the T6SS4 operon in Yersinia pseudotuberculosis to facilitate Zn2+ uptake and oxidative stress resistance. Zur regulates the expression of ZnuABC and T6SS4 by directly binding to their promoter regions. Zur senses the Zn2+ concentration and represses ZnuABC in a Zn2+-containing environment. Zur works as an auxiliary regular activator of T6SS4, facilitating oxidative stress resistance. This study revealed the dual function of regulator Zur on ZnuABC and T6SS4, and enriched the knowledge of Zn2+ homeostasis maintenance in Y. pseudotuberculosis.

Contact-independent killing mediated by a T6SS effector with intrinsic cell-entry properties.

Bacterial type VI secretion systems (T6SSs) inject toxic effectors into adjacent eukaryotic and prokaryotic cells. It is generally thought that this process requires physical contact between the two cells. Here, we provide evidence of contact-independent killing by a T6SS-secreted effector. We show that the pathogen Yersinia pseudotuberculosis uses a T6SS (T6SS-3) to secrete a nuclease effector that kills other bacteria in vitro and facilitates gut colonization in mice. The effector (Tce1) is a small protein that acts as a Ca2+- and Mg2+-dependent DNase, and its toxicity is inhibited by a cognate immunity protein, Tci1. As expected, T6SS-3 mediates canonical, contact-dependent killing by directly injecting Tce1 into adjacent cells. In addition, T6SS-3 also mediates killing of neighboring cells in the absence of cell-to-cell contact, by secreting Tce1 into the extracellular milieu. Efficient contact-independent entry of Tce1 into target cells requires proteins OmpF and BtuB in the outer membrane of target cells. The discovery of a contact-independent, long-range T6SS toxin delivery provides a new perspective for understanding the physiological roles of T6SS in competition. However, the mechanisms mediating contact-independent uptake of Tce1 by target cells remain unclear.

Citrus Physiological and Molecular Response to Boron Stresses.

Since the essentiality of boron (B) to plant growth was reported nearly one century ago, the implication of B in physiological performance, productivity and quality of agricultural products, and the morphogenesis of apical meristem in plants has widely been studied. B stresses (B deficiency and toxicity), which lead to atrophy of canopy and deterioration of Citrus fruits, have long been discovered in citrus orchards. This paper reviews the research progress of B stresses on Citrus growth, photosynthesis, light use efficiency, nutrient absorption, organic acid metabolism, sugar metabolism and relocation, and antioxidant system. Moreover, the beneficial effects of B on plant stress tolerance and further research in this area were also discussed.

QTLs identification for nitrogen and phosphorus uptake-related traits using ultra-high density SNP linkage.

To understand the genetic basis of nitrogen and phosphorus uptake in the cultivated rice, quantitative trait loci (QTL) analysis for 7 nitrogen and phosphorus uptake-related traits including above-ground biomass (AGB), leaf colour value (SPAD) in heading stage, grain nitrogen concentration (GNC), grain nitrogen content of the plant, total nitrogen content (TNC), grain phosphorus concentration, total phosphorus content (TPC) were conducted using SNP markers in a F2 population derived from a cross between GH128 and W6827. A total of 21 QTLs for nitrogen and phosphorus uptake-related traits distributed in 16 regions along 6 chromosomes were detected using a high density genetic map consisting of 1582 bin markers, with QTLs maximum explaining 8.19% of the phenotypic variation. Nine QTLs (42.9% of total QTLs) were detected on chromosome 2. Among them, two QTL clusters including AGB, TNC, TPC and GNC were also detected in the region bin 140 and bin 146 on the chromosome 2. The distance between the two clusters was only 4.1 cM. The presence of QTL clusters has important significance and could be useful in molecular marker assisted breeding. These genomic regions might be deployed for the simultaneous improving the use efficiency of nitrogen and phosphorus in rice breeding.

The stringent response factor, RelA, positively regulates T6SS4 expression through the RovM/RovA pathway in Yersinia pseudotuberculosis.

The type VI secretion system (T6SS) is a versatile molecular machinery widely distributed in Gram-negative bacteria. The activity of the T6SS is tightly regulated by various mechanisms, including quorum sensing (QS), iron concentration, and transcriptional regulators. Here we demonstrated that the stringent response regulator, RelA, contributes to bacterial resistance to multiple environmental stresses in Yersinia pseudotuberculosis. We also revealed that the stress resistance function of stringent response (SR) was partially mediated by the general stress response T6SS4 system. RelA positively regulates the expression of T6SS4 to combat various stresses in response to nutrition starvation collectively mediated by the RovM and RovA regulators. These findings revealed not only the important role of T6SS4 in SR induced stress resistance, but also a new pathway to regulate T6SS4 expression in response to starvation stress.

Myo-inositol-1-phosphate synthase (Ino-1) functions as a protection mechanism in Corynebacterium glutamicum under oxidative stress.

Reactive oxygen species (ROS) generated in aerobic metabolism and oxidative stress lead to macromolecules damage, such as to proteins, lipids, and DNA, which can be eliminated by the redox buffer mycothiol (AcCys-GlcN-Ins, MSH). Myo-inositol-phosphate synthase (Ino-1) catalyzes the first committed step in the synthesis of MSH, thus playing a critical role in the growth of the organism. Although Ino-1s have been systematically studied in eukaryotes, their physiological and biochemical functions remain largely unknown in bacteria. In this study, we report that Ino-1 plays an important role in oxidative stress resistance in the gram-positive Actinobacteria Corynebacterium glutamicum. Deletion of the ino-1 gene resulted in a decrease in cell viability, an increase in ROS production, and the aggravation of protein carbonylation levels under various stress conditions. The physiological roles of Ino-1 in the resistance to oxidative stresses were corroborated by the absence of MSH in the Δino-1 mutant. In addition, we found that the homologous expression of Ino-1 in C. glutamicum yielded a functionally active protein, while when expressed in Escherichia coliBL21(DE3), it lacked measurable activity. An examination of the molecular mass (Mr) suggested that Ino-1 expressed in E. coliBL21(DE3) was not folded in a catalytically competent conformation. Together, the results unequivocally showed that Ino-1 was important for the mediation of oxidative resistance by C. glutamicum.

Optimized nitrogen management enhances lodging resistance of rice and its morpho-anatomical, mechanical, and molecular mechanisms.

Increasing evidence shows that improved nitrogen management can enhance lodging resistance and lower internodes play a key role in the lodging resistance of rice. However, little is known about the cellular and molecular mechanisms underlying the enhanced lodging resistance under improved nitrogen management. In the present study, two rice varieties, with contrasting lodging resistance, were grown under optimized N management (OPT) and farmers' fertilizer practices. Under OPT, the lower internodes of both cultivars were shorter but the upper internodes were longer, while both culm diameter and wall thickness of lower internodes were dramatically increased. Microscopic examination showed that the culm wall of lower internodes under OPT contained more sclerenchyma cells beneath epidermis and vascular bundle sheath. The genome-wide gene expression profiling revealed that transcription of genes encoding cell wall loosening factors was down-regulated while transcription of genes participating in lignin and starch synthesis was up-regulated under OPT, resulting in inhibition of longitudinal growth, promotion in transverse growth of lower internodes and enhancement of lodging resistance. This is the first comprehensive report on the morpho-anatomical, mechanical, and molecular mechanisms of lodging resistance of rice under optimized N management.

Type VI Secretion Systems Present New Insights on Pathogenic Yersinia.

The type VI secretion system (T6SS) is a versatile secretion system widely distributed in Gram-negative bacteria that delivers multiple effector proteins into either prokaryotic or eukaryotic cells, or into the extracellular milieu. T6SS participates in various physiological processes including bacterial competition, host infection, and stress response. Three pathogenic Yersinia species, namely Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica, possess different copies of T6SSs with distinct biological functions. This review summarizes the pathogenic, antibacterial, and stress-resistant roles of T6SS in Yersinia and the ion-transporting ability in Y. pseudotuberculosis. In addition, the T6SS-related effectors and regulators identified in Yersinia are discussed.

Transcriptional control of the phenol hydroxylase gene phe of Corynebacterium glutamicum by the AraC-type regulator PheR.

Corynebacterium glutamicum can degrade phenol by a meta-cleavage pathway, which depends on ncgl2588 (phe) of the phe operon encoding phenol hydroxylase. An additional gene, ncgl2587 (pheR), is located upstream of phe. The pheR encodes an AraC/XylR-type regulator protein with 377 amino acid residues and is transcribed in the same direction as phe. Disruption of pheR by homologous recombination resulted in the accumulation of phenol in C. glutamicum. PheR demonstrates a low type of constitutive expression where phenol induces phe expression. PheR shares 75% sequence identity with AraC-type regulator of Corynebacterium lubricantis and 37 conserved residues, characteristic of AraC family, were located. A constructed pK18mobsacB-Pphe:lacZ transcriptional fusion plasmid was transformed into the wild-type, ΔpheR, and ΔpheR+ strains, and the results indicated that PheR activates the expression of phe encoding phenol hydroxylase. Electrophoretic mobility shift assay (EMSA) demonstrated a direct interaction of PheR with the phe promoter region and binding site of PheR on the Pphe was located 109-bp upstream of phe, as indicated by foot printing analysis. Our research provides deep insight into PheR expression and its regulatory function on Phe in C. glutamicum.