Dongqing Xu

Overexpression of a TFIIIA-type zinc finger protein gene ZFP252 enhances drought and salt tolerance in rice (Oryza sativa L.).

We previously identified a salt and drought stress‐responsive TFIIIA‐type zinc finger protein gene ZFP252 from rice. Here we report the functional analysis of ZFP252 using gain‐ and loss‐of‐function strategies. We found that overexpression of ZFP252 in rice increased the amount of free proline and soluble sugars, elevated the expression of stress defense genes and enhanced rice tolerance to salt and drought stresses, as compared with ZFP252 antisense and non‐transgenic plants. Our findings suggest that ZFP252 plays an important role in rice response to salt and drought stresses and is useful in engineering crop plants with enhanced tolerance to salt and drought stresses.

Increased tolerance of rice to cold, drought and oxidative stresses mediated by the overexpression of a gene that encodes the zinc finger protein ZFP245.

ZFP245 is a cold- and drought-responsive gene that encodes a zinc finger protein in rice. The ZFP245 protein localizes in the nucleus and exhibits trans-activation activity. Transgenic rice plants overexpressing ZFP245 were generated and found to display high tolerance to cold and drought stresses. The transgenic plants did not exhibit growth retardation, but showed growth sensitivity against exogenous abscisic acid, increased free proline levels and elevated expression of rice pyrroline-5-carboxylatesynthetase and proline transporter genes under stress conditions. Overproduction of ZFP245 enhanced the activities of reactive oxygen species-scavenging enzymes under stress conditions and increased the tolerance of rice seedlings to oxidative stress. Our data suggest that ZFP245 may contribute to the tolerance of rice plants to cold and drought stresses by regulating proline levels and reactive oxygen species-scavenging activities, and therefore may be useful for developing transgenic crops with enhanced tolerance to abiotic stress.

Expression analysis of rice A20/AN1-type zinc finger genes and characterization of ZFP177 that contributes to temperature stress tolerance

The A20/AN1-type zinc finger protein family is conserved in animals and plants. Using human AWP1 protein as a query, we identified twelve A20/AN1-type zinc finger proteins in japonica rice. Most of these genes were constitutively expressed in leaves, roots, culms and spikes. Through microarray analysis, it was found that four genes (ZFP177, ZFP181, ZFP176, ZFP173), two genes (ZFP181 and ZFP176) and one gene (ZFP157) were significantly induced by cold, drought and H(2)O(2) treatments, respectively. Further expression analysis showed that ZFP177 was responsive to both cold and heat stresses, but down-regulated by salt. The subcellular localization assay indicated that ZFP177 was localized in cytoplasm in tobacco leaf and root cells. Yeast-one hybrid assay showed that ZFP177 lacked trans-activation potential in yeast cells. Overexpression of ZFP177 in tobacco conferred tolerance of transgenic plants to both low and high temperature stresses, but increased sensitivity to salt and drought stresses. Further we found expression levels of some stress-related genes were inhibited in ZFP177 transgenic plants. These results suggested that ZFP177 might play crucial but differential roles in plant responses to various abiotic stresses.

A TFIIIA-type zinc finger protein confers multiple abiotic stress tolerances in transgenic rice (Oryza sativa L.).

The TFIIIA-type zinc finger transcription factors are involved in plant development and abiotic stress responses. Most TFIIIA-type zinc finger proteins are transcription repressors due to existence of an EAR-motif in their amino acid sequences. In this work, we found that ZFP182, a TFIIIA-type zinc finger protein, forms a homodimer in the nucleus and exhibits trans-activation activity in yeast cells. The deletion analysis indicated that a Leu-rich region at C-terminus is required for the trans-activation. Overexpression of ZFP182 significantly enhanced multiple abiotic stress tolerances, including salt, cold and drought tolerances in transgenic rice. Overexpression of ZFP182 promotes accumulation of compatible osmolytes, such as free proline and soluble sugars, in transgenic rice. ZFP182 activates the expression of OsP5CS encoding pyrroline-5-carboxylate synthetase and OsLEA3 under stress conditions, while OsDREB1A and OsDREB1B were regulated by ZFP182 under both normal and stress conditions. Interestingly, site-directed mutagenesis assay showed that DRE-like elements in ZFP182 promoter are involved in dehydration-induced expression of ZFP182. The yeast two-hybrid assay revealed that ZFP182 interacted with several ribosomal proteins including ZIURP1, an ubiquitin fused to ribosomal protein L40. The in vivo and in vitro interactions of ZFP182 and ZIURP1 were further confirmed by bimolecular fluorescence complementation and His pull-down assays. Our studies provide new clues in the understanding of the mechanisms for TFIIIA-type zinc finger transcription factor mediated stress tolerance and a candidate gene for improving stress tolerance in crops.

Comparative genome analyses of Serratia marcescens FS14 reveals its high antagonistic potential.

S. marcescens FS14 was isolated from an Atractylodes macrocephala Koidz plant that was infected by Fusarium oxysporum and showed symptoms of root rot. With the completion of the genome sequence of FS14, the first comprehensive comparative-genomic analysis of the Serratia genus was performed. Pan-genome and COG analyses showed that the majority of the conserved core genes are involved in basic cellular functions, while genomic factors such as prophages contribute considerably to genome diversity. Additionally, a Type I restriction-modification system, a Type III secretion system and tellurium resistance genes are found in only some Serratia species. Comparative analysis further identified that S. marcescens FS14 possesses multiple mechanisms for antagonism against other microorganisms, including the production of prodigiosin, bacteriocins, and multi-antibiotic resistant determinants as well as chitinases. The presence of two evolutionarily distinct Type VI secretion systems (T6SSs) in FS14 may provide further competitive advantages for FS14 against other microbes. To our knowledge, this is the first report of comparative analysis on T6SSs in the genus, which identifies four types of T6SSs in Serratia spp.. Competition bioassays of FS14 against the vital plant pathogenic bacterium Ralstonia solanacearum and fungi Fusarium oxysporum and Sclerotinia sclerotiorum were performed to support our genomic analyses, in which FS14 demonstrated high antagonistic activities against both bacterial and fungal phytopathogens.

Isolation of proteorhodopsin-bearing bacterium JL-3 from fresh water and characterization of the proteorhodopsin

Proteorhodopsins (PRs), light-driven proton pumps, constitute the largest family of the microbial rhodopsins. PRs are widely distributed in the oceanic environment and freshwater, but no bacteria with PRs have been isolated from freshwater so far. To facilitate isolation of the bacteria with PR genes, we constructed a vector system that can be used to clone potential PR genes and render color changes when overexpressed in Escherichia coli. Using this method, we successfully isolated a strain with PR gene from freshwater and identified it as Exiguobacterium sp. JL-3. The full length PR gene was then cloned using the SEFA PCR method. Protein sequence alignment showed that JL-3_PR shares high sequence identity (84-89%) with the PRs from Exiguobacterium strains, but low sequence identity (< 38%) with other PRs. Surprisingly, we could not detect any proton-pumping activity in the native JL-3 cells and protoplasts, but the recombinant JL-3_PR do pump protons when overexpressed in E. coli. Sequence analysis further revealed that the PRs from Exiguobacterium had an unusual lysine as the proton donor instead of the typical acidic residue. These data suggest that JL-3_PR is a sensory PR rather than a proton pump.

Identification of a toxic serralysin family protease with unique thermostable property from S. marcescens FS14

A serralysin family protease (Serralysin-like protease B, SPB) with unique V-shaped thermostable property was isolated and identified from the Serratia marcescens FS14 by biochemical and molecular biological methods. It is the first time to report the isolation of a native serralysin family protease directly from Serratia species except the well-studied serralysin. SPB has an optimum pH at 8.0 and an optimum temperature at 37°C. It shows high proteolytic activities after pretreated at 4-50°C for 10min respectively and almost no detectable activity after pretreated at 60°C. Surprisingly, increasing activities were observed after pretreated at 70-90°C respectively. Further study revealed that the reason behind this phenomenon may be the self-digestion property of SPB with an optimum temperature around 60°C. This self-digestion property may expand the SPB future application in industry. The bioassay using the healthy cotton bollworm Helicoverpa armigera larvae demonstrated that the serralysin and SPB from FS14 are toxic to the H. armigera larvae. This result implied that FS14 strain and/or the SPB and serralysin in FS14 might have a potential application in insect control.

Crystal Structure and Function of PqqF Protein in the Pyrroloquinoline Quinone Biosynthetic Pathway.

Pyrroloquinoline quinone (PQQ) has received considerable attention due to its numerous important physiological functions. PqqA is a precursor peptide of PQQ with two conserved residues: glutamate and tyrosine. After linkage of the Cγ of glutamate and Cϵ of tyrosine by PqqE, these two residues are hypothesized to be cleaved from PqqA by PqqF. The linked glutamate and tyrosine residues are then used to synthesize PQQ. Here, we demonstrated that the pqqF gene is essential for PQQ biosynthesis as deletion of it eliminated the inhibition of prodigiosin production by glucose. We further determined the crystal structure of PqqF, which has a closed clamshell-like shape. The PqqF consists of two halves composed of an N- and a C-terminal lobe. The PqqF-N and PqqF-C lobes form a chamber with the volume of the cavity of ∼9400 Å3. The PqqF structure conforms to the general structure of inverzincins. Compared with the most thoroughly characterized inverzincin insulin-degrading enzyme, the size of PqqF chamber is markedly smaller, which may define the specificity for its substrate PqqA. Furthermore, the 14-amino acid-residue-long tag formed by the N-terminal tag from expression vector precisely protrudes into the counterpart active site; this N-terminal tag occupies the active site and stabilizes the closed, inactive conformation. His-48, His-52, Glu-129 and His-14 from the N-terminal tag coordinate with the zinc ion. Glu-51 acts as a base catalyst. The observed histidine residue-mediated inhibition may be applicable for the design of a peptide for the inhibition of M16 metalloproteases.

Expression, crystallization and preliminary crystallographic data analysis of VioD, a hydroxylase in the violacein-biosynthesis pathway.

Violacein, a natural purple secondary metabolite, is sequentially biosynthesized by five enzymes in the following pathway: VioA-VioB-VioE-VioD-VioC. VioD, a flavin-dependent oxygenase, catalyzes the hydroxylation of the intermediate product prodeoxyviolaceinic acid (PVA) at the 5-position of one indole ring to yield proviolacein. In vitro biochemical data have revealed this process, but the catalytic mechanism still remains largely unclear. Here, the cloning, expression, purification, crystallization and diffraction of VioD are reported. Crystals of VioD diffracted to 1.7 Å resolution and belonged to space group P31, with unit-cell parameters a = b = 90.0, c = 94.5 Å, α = β = 90, γ = 120°. Solvent-content calculation and molecular-replacement results suggest the presence of two molecules of VioD in the asymmetric unit.

Expression, purification, crystallization and preliminary crystallographic analysis of Cg1458: a novel oxaloacetate decarboxylase from Corynebacterium glutamicum.

Oxaloacetate decarboxylase catalyses the decarboxylation of oxaloacetate to pyruvate and CO(2). Recently, the Corynebacterium glutamicum gene product Cg1458 was determined to be a soluble oxaloacetate decarboxylase. To elucidate the mechanism of oxaloacetate decarboxylation by Cg1458, recombinant Cg1458 was purified and crystallized. The best crystal was grown from 0.2 M MgCl(2), 0.1 M Bis-Tris pH 6.0, 25%(w/v) polyethylene glycol 3350 using the hanging-drop method. The crystals belonged to space group P4(3)2(1)2, with unit-cell parameters a = b = 124.1, c = 73.6 Å. The crystals are most likely to contain a dimer in the asymmetric unit, with a V(M) value of 2.27 Å(3) Da(-1). A full data set was collected at 1.9 Å resolution using synchrotron radiation on beamline BL17U of SSRF, Shanghai, China. Structure-solution attempts by molecular replacement were successful with PDB entries 3qdf or 2dfu as the template.