Improvement of uridine production in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> by metabolic engineering

Objectives

To construct a Bacillus subtilis strain for improved uridine production.

Results

The AAG_2846–2848 fragment of the pyrAB gene, encoding carbamoylphosphate synthetase, was deleted in B. subtilis TD246 leading to a 245% increase of uridine production and the conversion from glucose to uridine increased by 10.5%. Overexpression of the pyr operon increased the production of uridine by a further 31% and the conversion rate of glucose to uridine was increased by 18%. In addition, the blocking of arginine synthesis or disabling of glutamate dehydrogenase significantly enhanced the uridine production. The highest-producing strain, B. subtilis TD297, accumulated 11 g uridine/l with a yield of 240 mg uridine/g glucose in shake-flask cultivation.

Conclusion

This is the first report of engineered B. subtilis strains which can produce more than 11 g uridine/l, with a yield reaching 240 mg uridine/g glucose in shake-flask cultivation.

     

Polymethylene derivatives of nucleic bases with ω-functional groups: VI. [8-(2-oxocyclohexyl)-9-oxooctyl]pyrimidines as potential inhibitors of pyrimidine phosphorylases

New polymethylene derivatives of nucleic bases with β-diketo function in ω-position were prepared by alkylation of uracil, thymine, and cytosine. Their physicochemical properties and effect on the E. coli uridine and thimidine phosphorylases were studied.     

Activities and properties of phosphorylases of turbellarias <Emphasis Type="Italic">Phagocata sibirica</Emphasis> and cestodes <Emphasis Type="Italic">Bothriocephalus scorpii</Emphasis>

Activities and properties of phosphorylases of cytosol and mitochondrial fractions are studied in free-living turbellarias Phagocata sibirica and cestodes Bothriocephalus scorpii. The phosphorylase activities in P. sibirica and B. scorpii differ significantly both in form and in total activity of this enzyme. Dependence of the phosphorylase reaction rate on substrate concentration is studied. The high activity of phosphorylase as compared with that of hexokinase suggests glycogen to be the main energy source of the studied flatworms. Action of various effectors on activities of the cytosol and mitochondrial phosphorylases has been studied.     

The effects of gibberellins and mepiquat chloride on nitrogenase activity in <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis>

Application of plant growth regulators (PGRs) to soybean plants is known to induce changes in nitrogenase activity in root nodules, and this led us to hypothesize that PGRs would affect nitrogenase activity in free-living rhizobia cultures. Little is known about the molecular basis of the effects of PGRs on nitrogenase activity in free-living rhizobia cultures. Therefore, a comparative study was conducted on the effects of gibberellins (GA₃) and mepiquat chloride (PIX), which regulate plant growth, on the nitrogenase activity of the nitrogen-fixing bacterium Bradyrhizobium japonicum. Fix and nif gene regulation and protein expression in free-living cultures of B. japonicum were investigated using real-time PCR and two-dimensional electrophoresis after treatment with GA₃ or PIX. GA₃ treatment decreased nitrogenase activity and the relative expression of nifA, nifH, and fixA genes, but these effects were reversed by PIX treatment. As expected, several proteins involved in nitrogenase synthesis were down-regulated in the GA₃-treated group. Conversely, several proteins involved in nitrogenase synthesis were up-regulated in the PIX-treated group, including bifunctional ornithine acetyltransferase/N-acetylglutamate synthase, transaldolase, ubiquinol-cytochrome C reductase iron-sulfur subunit, electron transfer flavoprotein subunit beta, and acyl-CoA dehydrogenase. Two-pot experiments were conducted to evaluate the effects of GA₃ and PIX on nodulation and nitrogenase activity in Rhizobium-treated legumes. Interestingly, GA₃ treatment increased nodulation and depressed nitrogenase activity, but PIX treatment decreased nodulation and enhanced nitrogenase activity. Our data show that the nif and fix genes, as well as several proteins involved in nitrogenase synthesis, are up-regulated by PIX and down-regulated by GA₃, respectively, in B. japonicum.     

Generation of Chinese cabbage resistant to bacterial soft rot by heterologous expression of <Emphasis Type="Italic">Arabidopsis WRKY75</Emphasis>

Soft rot caused by Pectobacterium carotovorum subsp. carotovorum (Pcc) is a serious disease in Chinese cabbage (Brassica rapa L. subsp. pekinensis). To reduce the severity of soft rot symptoms in Chinese cabbage, Arabidopsis AtWRKY75 was introduced into Chinese cabbage by Agrobacterium-mediated transformation, which was previously reported to reduce susceptibility to Pcc infection in Arabidopsis. Three independent Chinese cabbage transgenic lines carrying AtWRKY75 were obtained. The growth phenotypes of AtWRKY75 overexpression (OE) lines were normal. Bacterial soft rot symptoms and Pcc growth were reduced in AtWRKY75-OE Chinese cabbage lines compared with WT plants. In contrast, overexpression of AtWRKY75 had no effect on infection with a hemibiotrophic pathogen, Xanthomonas campestris pv. campestris (Xcc) causing black rot disease. These results are consistent with those observed in the transgenic Arabidopsis. We found that AtWRKY75 activated a subset of Chinese cabbage genes related to defense against Pcc infection, such as Meri15B, BrPR4, and BrPDF1.2 (but not BrPGIP2). Moreover, overexpression of AtWRKY75 caused H₂O₂ production and activation of H₂O₂ scavenge enzyme genes, suggesting that H₂O₂ played a role in AtWRKY75-mediated resistance to Pcc. Together, these results demonstrated that AtWRKY75 decreased the severity of Pcc-caused bacterial soft rot and activated a subset of Pcc infection defense-related genes in Chinese cabbage similar to in Arabidopsis. It is suggested that AtWRKY75 is a candidate gene for use in crop improvement, because it results in reduced severity of disease symptoms without concurrent growth abnormalities.     

Molecular characterization and subcellular localization of salt-inducible lipid transfer proteins in rice

Rice (Oryza sativa L.) is a salt-sensitive species. Salt stress can cause injury to the plant cellular membrane. Plant lipid transfer proteins (LTPs) are abundant lipid binding proteins that are important in membrane vesicle biogenesis and trafficking, however, the biological importance of LTPs on salt-stress response in rice remains unclear. Therefore, salt-responsive rice LTPs were identified and characterized in this study. Microarray analysis showed seven genes positively regulated by salinity, including five Ltp genes (LtpII.3, LtpII.5, LtpII.6, LtpV.1, and LtpV.2) and two Ltp-like (LtpL; LtpL1, and LtpL2) genes. Amino acid alignment revealed that all these Ltp and LtpL genes contained the N-terminal signal peptide. Apart from LtpL1, all salt-inducible Ltp genes had the conserved eight cysteine residue motifs backbone. Verification of gene expression to different stimuli in rice seedlings revealed that salt-regulated Ltp genes differentially responded to drought, cold, H₂O₂, abscisic acid (ABA) and CaCl₂. Furthermore, the expression of Ltp and LtpL genes was tissue-specifically regulated by ABA-dependent and independent pathway. In silico analysis of a 1.5-kb 5’-upstream region of these genes showed regulatory cis-elements associated with ABA, calcium, and cold/drought responses. Three LtpII subfamily genes, including LtpII.3, LtpII.5, and LtpII.6, were strictly expressed in flowers and seeds, and LtpIII.1 mRNA strongly accumulated in stem tissue. Subcellular localization analysis of LTP-DsRed fusion proteins revealed that the five LTPs and two LTPLs localized at the endoplasmic reticulum. The results provide new clues to further understanding the biological functions of Ltp genes.     

Selection and Characterization of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> (Berliner) (Eubacteriales: Bacillaceae) Strains for <Emphasis Type="Italic">Ecdytolopha aurantiana</Emphasis> (Lima) (Lepidoptera: Tortricidae) Control

The citrus fruit borer, Ecdytolopha aurantiana (Lima, 1927) (Lepidoptera: Tortricidae), is responsible for major losses to the citrus industry because it causes rot and drop of fruits. The current study aimed to select and characterize Bacillus thuringiensis (Berliner, 1911) strains toxic to E. aurantiana. For this purpose, 47 B. thuringiensis strains were evaluated in selective bioassays using first instar larvae of E. aurantiana. The lethal concentration (LC₅₀) of the most toxic strains was estimated, and the strains were characterized by morphological, biochemical, and molecular methods. Of the 47 strains tested, 10 caused mortality above 85% and showed mean lethal concentrations between 1.05E+7 and 1.54E+8 spores mL^?1. The lowest LC₅₀ values were obtained for the HD-1 standard strain and the BR145, BR83, BR52, and BR09 strains. The protein profile showed the presence of Cry proteins of 60, 65, 70, 80, and 130 kDa. The molecular characterization showed the presence of cry1, cry2, cry3, and cry11 genes. The morphological analysis identified three different crystalline inclusions: bipyramidal, round, and cuboidal. The cry1 and cry2 genes were the most frequent among the B. thuringiensis strains evaluated and encode Cry proteins toxic to insects of the order Lepidoptera, which agree with the toxicity results obtained by the selective bioassays against E. aurantiana. The results showed four different B. thuringiensis strains toxic to E. aurantiana at the same level as the HD-1 standard strain, and these strains have biotechnological potential for E. aurantiana control through the production of transgenic plants or the formulation of biopesticides.     

The mathematical model of concentration polarization coefficient in membrane transport and volume flows

In this paper, the authors investigate the membrane transport of aqueous non-electrolyte solutions in a single-membrane system with the membrane mounted horizontally. The purpose of the research is to analyze the influence of volume flows on the process of forming concentration boundary layers (CBLs). A mathematical model is provided to calculate dependences of a concentration polarization coefficient (ζ _s ) on a volume flux (J _vm ), an osmotic force (Δπ) and a hydrostatic force (ΔP) of different values. Property ζ _s ?=?f(J _vm ) for J _vm ?>?0 and for J _vm ?≈?0 and property ζ _s ?=?f(ΔC ₁) are calculated. Moreover, results of a simultaneous influence of ΔP and Δπ on a value of coefficient ζ _s when J _vm ?=?0 and J _vm ?≠?0 are investigated and a graphical representation of the dependences obtained in the research is provided. Also, mathematical relationships between the coefficient ζ _s and a concentration Rayleigh number (R _C ) were studied providing a relevant graphical representation. In an experimental test, aqueous solutions of glucose and ethanol were used.     

Comparative Analysis of Mono- and Bifunctional Chorismate Synthases in <Emphasis Type="Italic">Escherichia coli</Emphasis> Cells Capable and Incapable of Phenylalanine Production

The activity of chorismate synthase, the terminal enzyme of the common aromatic pathway, is absolutely dependent on reduced flavin mononucleotide. The bifunctional chorismate synthase of Saccharomyces cerevisiae (product of the ARO2 gene) can reduce flavin in a reaction that involves NADPH, in contrast to the monofunctional chorismate synthase of Escherichia coli (product of the _aro C gene). The latter enzyme does not have the capacity for flavin reduction, and its activity therefore depends on the flavin reductase function of the cell. Chemical synthesis of the structural part of the ARO2 gene that involved the substitution of rare E. coli codons was performed for an in vivo comparison of the two types of chorismate synthase. ARO2 expression was tested in the T7 system, and isogenic E. coli strains TG1Δ _aro CP_tac-ARO2 and TG1Δ _aro CP_tac- _aro C were obtained. Comparative analysis of proteins from the cell extracts of these strains and in silico assessment of hybrid RBS efficiency showed that the level of AroC protein synthesis in TG1Δ _aro CP_tac- _aro C was higher than the level of ARO2 synthesis in the TG1Δ _aro CP_tac-ARO2 cells. The introduction of Ptac-ARO2 and Ptac- _aro C modifications led to complete recovery of the growth of the aromatic auxotroph TG1Δ _aro C on minimal mineral medium supplemented with glucose and restored phenylalanine production in the E. coli strain DV1017Δ _aro C, which lacked chorismate synthase activity. The similar positive effects of P_tac- _aro C and P_tac-ARO2 on phenylalanine biosynthesis in the DV1017ΔtyrR strain, in which chorismate synthase played a “bottleneck” role, indicated the absence of a limiting effect of reduced flavin on monofunctional chorismate synthase overexpressed in E. coli cells.     

Over-expression of the <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> harpin-encoding gene <Emphasis Type="Italic">hrpZm</Emphasis> confers enhanced tolerance to Phytophthora root and stem rot in transgenic soybean

Phytophthora root and stem rot (PRR) caused by Phytophthora sojae is one of the most devastating diseases reducing soybean (Glycine max) production all over the world. Harpin proteins in many plant pathogenic bacteria were confirmed to enhance disease and insect resistance in crop plants. Here, a harpin protein-encoding gene hrpZpsta from the P. syringae pv. tabaci strain Psta218 was codon-optimized (renamed hrpZm) and introduced into soybean cultivars Williams 82 and Shennong 9 by Agrobacterium-mediated transformation. Three independent transgenic lines over-expressing hrpZm were obtained and exhibited stable and enhanced tolerance to P. sojae infection in T₂–T₄ generations compared to the non-transformed (NT) and empty vector (EV)-transformed plants. Quantitative real-time PCR (qRT-PCR) analysis revealed that the expression of salicylic acid-dependent genes PR1, PR12, and PAL, jasmonic acid-dependent gene PPO, and hypersensitive response (HR)-related genes GmNPR1 and RAR was significantly up-regulated after P. sojae inoculation. Moreover, the activities of defense-related enzymes such as phenylalanine ammonia lyase (PAL), polyphenoloxidase (PPO), peroxidase, and superoxide dismutase also increased significantly in the transgenic lines compared to the NT and EV-transformed plants after inoculation. Our results suggest that over-expression of the hrpZm gene significantly enhances PRR tolerance in soybean by eliciting resistance responses mediated by multiple defense signaling pathways, thus providing an alternative approach for development of soybean varieties with improved tolerance against the soil-borne pathogen PRR.     

Secretion of Extracellular Proteinases Active against Fibrillar Proteins by Micromycetes

It has been shown that micromycetes Aspergillus ustus 1 and Tolypocladium inflatum k1 secrete proteolytic enzymes that possess high collagenolytic, fibrinolytic, and elastolytic activity. The activity of proteinases hydrolyzing fibrillar proteins, which was determined by the cleavage of azo-collagen, was 122.6 × 10^–3E_Azc/mL in A. ustus 1 and 69.7 × 10^–3E_Azc/mL in T. inflatum k1 (E_Azc is the amount of azocollagen cleaved in 1 min (μg). The maximum values of activity were observed during submerged cultivation of A. ustus 1 for 4 days and of T. inflatum k1 for 5 days. It has been shown that the maximum of collagenolytic and general proteolytic activity during the cultivation of A. ustus 1 are time-separated, unlike T. inflatum k1, which, presumably, can simplify the procedure for obtaining proteinases active against fibrillar proteins.     

Evaluation of ethanol production and bioadsorption of heavy metals by various red seaweeds

The objective of this study was to evaluate ethanol production and bioadsorption with four red seaweeds, Gelidium amansii, Gracilaria verrucosa, Kappaphycus alvarezii and Eucheuma denticulatum. To produce ethanol, thermal acid hydrolysis, enzymatic saccharification and fermentation was carried out. After pretreatment, 38.5, 39.9, 31.0 and 27.5 g/L of monosaccharides were obtained from G. amansii, G. verrucosa, K. alvarezii and E. denticulatum, respectively. Ethanol fermentation was performed with Saccharomyces cerevisiae KCCM 1129 adapted to 80 g/L galactose. The ethanol productions by G. amansii, G. verrucosa, K. alvarezii and E. denticulatum were 18.8 g/L with Y _EtOH = 0.49, 19.1 g/L with Y _EtOH = 0.48, 14.5 g/L with Y _EtOH = 0.47 and 13.0 g/L with Y _EtOH = 0.47, respectively. The waste seaweed slurries after the ethanol fermentation were reused to adsorb Cd(II), Pb(II) and Cu(II). Using langmuir isotherm model, Cu(II) had the highest affinity for waste seaweeds with the highest q _max and electronegativity values among three heavy metals.     

The effects of brown alga proteins and polysaccharides on egg fertilization and the development of embryos of the sea urchin Strongylocentrotus intermedius A. Agassiz, 1863

The toxic effects of a water–ethanol extract of the brown alga Saccharina (= Laminaria) cichorioides and the isolated I₁ and I₂ proteins on egg fertilization and the development of embryos of the sea urchin Strongylocentrotus intermedius were revealed and studied. The protective effects of fucoidans and 1,3;1,6-β-D-glucans (laminarans, translam, and glucan II) that were isolated from the brown algae S. cichorioides and Fucus evanescens against the toxic impacts of the I₁ and I₂ proteins were examined. It was found that the lifespan of sea urchin embryos increased from 1.5 to 3 times in the presence of 1,3;1,6-β-D-glucans and fucoidans, whereas the I₁ and I₂ proteins decreased embryo viability by 2 times. Preadministration of polysaccharides completely neutralized the inhibitory effects of the proteins on developing embryos. It was also shown that a water–ethanol extract of S. cichorioides, derived I₁ and I₂ proteins, and fucoidans has a contraceptive effect.     

Comparative muscle proteomics/phosphoproteomics analysis provides new insight for the biosafety evaluation of <Emphasis Type="Italic">fat</Emphasis>-<Emphasis Type="Italic">1</Emphasis> transgenic cattle

The biosafety of fat-1 transgenic cattle has been a focus of our studies since the first fat-1 transgenic cow was born. In this study, we used tandem mass tag labeling, TiO₂ enrichment, and nanoscale liquid chromatography coupled with tandem mass spectrometry (nanol LC–MS/MS) to compare proteomic and phosphoproteomic profiling analyses of muscle between fat-1 transgenic cows and wild-type cows. A total of 1555 proteins and 900 phosphorylation sites in 159 phosphoproteins were identified in the profiling assessments, but only four differentially expressed proteins and nine differentially expressed phosphopeptides were detected in fat-1 transgenic cows relative to wild-type cows. Bioinformatics analyses showed that all of the identified proteins and phosphoproteins were mainly related to the metabolic processes of three major nutrients: carbohydrates, lipids, and proteins. All of these differentially expressed proteins might take part in DNA recombination, repair, and regulation of the immune system. In conclusion, most of the identified proteins and phosphoproteins exhibited few changes. Our results provide new insights into the biosafety of fat-1 transgenic cattle.     

Association of polymorphic markers of chemokine genes,their receptors,and <Emphasis Type="Italic">CD14</Emphasis> gene with coronary atherosclerosis

Atherosclerosis represents an inflammatory response to the disturbance of the endothelial layer in the arterial bloodstream. In the present study, an analysis of associations of polymorphic markers for the genes controlling synthesis of proteins involved in atherosclerosis pathogenesis in coronary atherosclerosis (CA) patients (217 subjects) and in a control group (250 subjects) was conducted. The following genes were examined: rs991804 (CCL2 gene), rs1126579 (CXCR2 gene), rs4074 (CXCL1 gene), rs4073 (CXCL8 gene), rs333 (CCR5 gene), rs2471859 (CXCR4 gene), rs1801157 (CXCL12 gene), and rs2569190 (CD14 gene). Using the Monte Carlo and Markov chain (APSampler) method, allele/genotype combinations associated with both low and high CA risk were revealed. The most important findings included the following: CXCR4*T/T + CCL2*C + CCR5*I/I (P_perm = 1 × 10^–6, OR = 0.44, 95% CI 0.3–0.63), CXCR2*C + CD14*C + CXCL12*G + CCL2*C + CCR5*D (P_perm = 4 × 10^–6, OR = 5.78, 95% CI 2.34–14.28), CD14*C + CCL2*C/C + CCR5*D (P_perm = 6.3 × 10^–6, OR = 5.81, 95% CI 2.17–15.56), CXCL8*A + CXCR2*C + CD14*T + CXCR4*C (P_perm = 0.01, OR = 3.21, 95% CI 1.63–6.31).     

Informative Value of Temperatures in Different Areas of the Human Body for Correcting Body Thermal Imbalance during Extravehicular Activities

Identification of noninvasive and informative sites on the body reflecting the development of body thermal imbalance during extravehicular activities (EVAs) is highly important for enhancing astronaut safety. Temperature changes were evaluated on several areas of the head (the mastoid fossa (T _mf ), the forehead (T _fo ), and the cheek (T _ch )) and on the fingers (T _fing ). Subjects were dressed in a multicompartment liquid cooling/warming garment. Studies I and II consisted of different combinations of hood versus garment cooling or warming imposed across stages; studies III and IV involved sagittally divided cooling or warming regimes with the hood worn (study III) or with the head uncovered (study IV). In studies I and II, T _mf significantly (P < 0.05) differed between stages 2, when the head was cooled and the rest of the body heated, and 3, when the head was heated and the rest of the body cooled. The T _mf changes were consistent with the thermal conditions imposed on the head but not reflective of the developing body heat deficit. In study III, the T _mf at stages 2 and 3 on the right or the left followed the thermal conditions on the ipsilateral side of the body (P < 0.01). In study IV, T _fing showed no significant differences across stages. In studies I–IV, T _fing showed consistent changes across stages (P < 0.05), reflecting the developing body heat deficit. In all studies, there were no significant differences in rectal temperature (T _re ) across stages. T _mf and temperatures at other head skin sites did not respond in accordance with the actual intensity of a heat or cold flux from the garment and were not reflective of the overall development of body thermal imbalance. T _fing was a more adequate indicator of initial thermal destabilization and provided information that would be useful for monitoring the thermal balance and comfort during EVAs.