Dual Positive Feedback Regulation of Protein Degradation of an Extra-cytoplasmic Function σ Factor for Cell Differentiation in Streptomyces coelicolor

Here we report that in Streptomyces coelicolor, the protein stability of an ECF σ factor SigT, which is involved in the negative regulation of cell differentiation, was completely dependent on its cognate anti-σ factor RstA. The degradation of RstA caused a ClpP/SsrA-dependent degradation of SigT during cell differentiation. This was consistent with the delayed morphological development or secondary metabolism in the ΔclpP background after rstA deletion or sigT overexpression. Meanwhile, SigT negatively regulated clpP/ssrA expression by directly binding to the clpP promoter (clpPp). The SigT-clpPp interaction could be disrupted by secondary metabolites, giving rise to the stabilized SigT protein and retarded morphological development in a non-antibiotic-producing mutant. Thus a novel regulatory mechanism was revealed that the protein degradation of the ECF σ factor was initiated by the degradation of its anti-σ factor, and was accelerated in a dual positive feedback manner, through regulation by secondary metabolites, to promote rapid and irreversible development of the secondary metabolism. This ingenious cooperation of intracellular components can ensure economical and exquisite control of the ECF σ factor protein level for the proper cell differentiation in Streptomyces.     

Proteasome involvement in a complex cascade mediating SigT degradation during differentiation of Streptomyces coelicolor

In Streptomyces coelicolor, the ECF sigma factor SigT negatively regulates cell differentiation, and is degraded by ClpP protease in a dual positive feedback manner. Here we further report that the proteasome is required for degradation of SigT, but not for degradation of its anti-sigma factor RstA, and RstA can protect SigT from degradation independent of the proteasome. Meanwhile, deletion of the proteasome showed reduced production of secondary metabolites, and the fermentation medium from wild type could promote SigT degradation. Furthermore, overexpression of redD or actII-orf4 in the proteasome-deficiency mutant resulted in SigT degradation and over-production of both undecylprodigiosin and actinorhodin. Therefore the proteasome is required for SigT degradation by affecting the production of secondary metabolites during cell differentiation.     

The effect of mutation at M307 in <Emphasis Type="Italic">FUT1</Emphasis> gene on susceptibility of <Emphasis Type="Italic">Escherichia coli</Emphasis> F18 and gene expression in Sutai piglets

Escherichia coli F18 (ECF18) is a common porcine enteric pathogen. The pathogenicity of ECF18 bacteria depends on the existence of ECF18 receptor in the brush border membranes of piglet’s small intestinal mucosa. Alpha (1) fucosyltransferase gene (FUT1) has been identified as the candidate gene controlling the adhesion to ECF18 receptor. The genetic variations in the position of M307 nucleotide in open reading frame of FUT1 have been proposed as a marker for selecting resistant pigs. The piglets were divided into three groups, AA, AG and GG, according to the genotypes present at M307 of FUT1. Small intestinal epithelium cells of piglets with AA, AG and GG genotypes were selected to test the adhesion capability of the wild type E.coli expressing F18ab fimbriae, the recombinant E. coli expressing F18ac fimbriae or the recombinant E. coli secreting and surface-displaying the FedF subunit of F18ab fimbriae, respectively. Here, we examined the distribution and expression of porcine FUT1 mRNA in different tissues in Sutai pigs using real-time PCR. The results showed that piglets with AA genotype show resistance, whereas piglets with GG or AG genotypes are sensitive to the pathogenic E. coli F18 in Sutai piglets. FUT1 was expressed in all the tissues that were examined, and the gene’s expression was highest in the lungs. There was no significant difference in expression level among the three genotypes in the liver, lung, stomach and duodenum, where the gene expression was relatively high. The present analysis suggested that mutation at M307 in FUT1 gene determines susceptibility of small intestinal epithelium to E. coli F18 adhesion in Sutai piglet and the expression of FUT1 gene may be regulated by other factors or the mutation was likely to be in linkage disequilibrium with some cis-regulatory variants.     

Inactivation of the extracytoplasmic function sigma factor Sig6 stimulates avermectin production in <Emphasis Type="Italic">Streptomyces avermitilis</Emphasis>

The role of the extracytoplasmic function (ECF) σ factor Sig6 (SAV663) in avermectin production by Streptomyces avermitilis was investigated by gene-deletion, complementation and over-expression experiments. Inactivation of Sig6 had no major effect on growth, stress responses, or morphology. Avermectin yield was increased 2- to 2.7-fold (~680 μg/ml) relative to the wild-type strain by deletion of the sig6 gene, and was restored to the wild-type level by introduction of a single copy of sig6. Introduction of extra multi-copy or integrative sig6 vectors into the wild-type decreased avermectin yield by 56–63%. Taken together, these findings indicate that Sig6 plays a negative regulatory role in avermectin production in S. avermitilis. RT-PCR analysis demonstrated that this role of Sig6 is mediated by the pathway-specific activator gene aveR.     

Changes in antioxidant gene expression and induction of oxidative stress in pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.) under Al stress

Aluminium toxicity has been recognized as a primary growth-limiting factor in acid soil, resulting in a decrease in plant growth and production. In this experiment we have studied the induction of oxidative stress and changes in antioxidant gene expression in pea (var. ALASKA) under aluminium (Al) stress. We have found that Al treatment affected the growth of pea plant and induced oxidative stress with a change in antioxidant gene expression profile. While the expression of glutathione-s-transferase (GST) and catalase (CAT) was more in root, cytosolic Ascorbate peroxidase (cAPX) expression increased in shoot under aluminium stress. Copper- Zinc Superoxide dismutase (Cu-Zn SOD) gene expression was higher after 24 h but decreased after 48 h along with elevated expression of manganese superoxide dismutase (MnSOD) and iron-superoxide dismutase (FeSOD) at higher aluminium contentrations after 24 and 48 h. Aluminium stress elevated hydrogen peroxide (H₂O₂) level and affected the growth. The proline content did not change significantly, whereas glutathione content increased with a decreased ascorbate content under Al stress. The present study indicates that aluminium treatment affected the antioxidant gene expression and induced oxidative stress in pea plant.     

Evaluation of electro‐coagulation–flocculation for harvesting marine and freshwater microalgae

Although microalgae are considered as a promising feedstock for biofuels, the energy efficiency of the production process needs to be significantly improved. Due to their small size and low concentration in the culture medium, cost‐efficient harvesting of microalgae is a major challenge. In this study, the use of electro‐coagulation–flocculation (ECF) as a method for harvesting a freshwater (Chlorella vulgaris) and a marine (Phaeodactylum tricornutum) microalgal species is evaluated. ECF was shown to be more efficient using an aluminum anode than using an iron anode. Furthermore, it could be concluded that the efficiency of the ECF process can be substantially improved by reducing the initial pH and by increasing the turbulence in the microalgal suspension. Although higher current densities resulted in a more rapid flocculation of the microalgal suspension, power consumption, expressed per kg of microalgae harvested, and release of aluminum were lower when a lower current density was used. The aluminum content of the harvested microalgal biomass was less than 1% while the aluminum concentration in the process water was below 2 mg L⁻¹. Under optimal conditions, power consumption of the ECF process was around 2 kWh kg⁻¹ of microalgal biomass harvested for Chlorella vulgaris and ca. 0.3 kWh kg⁻¹ for Phaeodactylum tricornutum. Compared to centrifugation, ECF is thus more energy efficient. Because of the lower power consumption of ECF in seawater, ECF is a particularly attractive method for harvesting marine microalgae. Biotechnol. Bioeng. 2011;108: 2320–2329. © 2011 Wiley Periodicals, Inc.     

Validation of reference genes for RT-qPCR normalization in common bean during biotic and abiotic stresses

Selection of reference genes is an essential consideration to increase the precision and quality of relative expression analysis by the quantitative RT-PCR method. The stability of eight expressed sequence tags was evaluated to define potential reference genes to study the differential expression of common bean target genes under biotic (incompatible interaction between common bean and fungus Colletotrichum lindemuthianum) and abiotic (drought; salinity; cold temperature) stresses. The efficiency of amplification curves and quantification cycle (C _q) were determined using LinRegPCR software. The stability of the candidate reference genes was obtained using geNorm and NormFinder software, whereas the normalization of differential expression of target genes [beta-1,3-glucanase 1 (BG1) gene for biotic stress and dehydration responsive element binding (DREB) gene for abiotic stress] was defined by REST software. High stability was obtained for insulin degrading enzyme (IDE), actin-11 (Act11), unknown 1 (Ukn1) and unknown 2 (Ukn2) genes during biotic stress, and for SKP1/ASK-interacting protein 16 (Skip16), Act11, Tubulin beta-8 (β-Tub8) and Unk1 genes under abiotic stresses. However, IDE and Act11 were indicated as the best combination of reference genes for biotic stress analysis, whereas the Skip16 and Act11 genes were the best combination to study abiotic stress. These genes should be useful in the normalization of gene expression by RT-PCR analysis in common bean, the most important edible legume.     

Biochemical, ECF18R, and RYR1 Gene Polymorphisms and Their Associations with Osteochondral Diseases and Production Traits in Pigs

This study reports the association of five blood types, three enzymes, two proteins, Escherichia coli F18 receptor gene (ECF18R), and the Ryanodin receptor (RYR1) gene with six production traits, four meat quality traits, and two osteochondral diseases in Swiss pig populations. Data on on-farm traits (daily weight gain, percent premium cuts, and backfat) and on station-tested traits (daily weight gain, feed conversion ratio, meat quality, and osteochondral lesions) were available on 3,918 and 303 animals, respectively. A mixed linear model with allele substitution effects was used for each trait by marker analysis (144 analyses). Significant marker-trait associations and allele substitution effects are presented. In general, heritability estimates for production and meat quality traits were higher than those for osteochondral lesions. Blood types lack significant associations with many traits except H and S types. Enzymes (mainly, glucose phosphate isomerase) and protein polymorphisms show significant associations with daily weight gain, premium cuts, and backfat as well as osteochondral lesions. The RYR and ECF18R genes significantly affected all growth, production, and lean meat content traits and osteochondral lesions; RYR also affected pH values. This study reports many novel marker-trait associations, particularly between the incidence of osteochondral lesions and polymorphisms at glucose phosphate isomerase, 6-phosphogluconate dehydrogenase, postalbumin 1A, RYR, and ECF18R loci. These results should be useful in selection and for further functional genomics and proteomics investigations.     

Endometriotic cyst fluid induces reactive oxygen species (ROS) in human immortalized epithelial cells derived from ovarian endometrioma

Objectives: Endometriotic cyst fluid (ECF) contains a large amount of reactive oxygen species (ROS), and endometriotic cysts are exposed to strong oxidative stress, which may cause malignant transformation. In this study, ROS production by ECF was clinically analysed.

Methods: Human immortalized epithelial cells derived from ovarian endometrioma (EMosis-CC/TERT 1) were treated with ECF. In addition, ROS production in EMosis-CC/TERT 1 was measured, and its clinical significance was analysed.

Results: A total of 38 ECF samples were obtained from patients diagnosed with endometriotic cysts. In EMosis-CC/TERT1, significantly higher levels of ROS were induced by ECF than by the vehicle control and ferric nitrilotriacetate. There were no significant differences in ROS production by laterality and preoperative serum CA125 values. There were several patients whose cyst sizes were approximately 5?cm and had relatively high ROS production. Production of ROS by ECF was relatively higher in patients older than 40 years of age than in those younger than 40.

Discussion: Our study revealed that ROS are highly produced by ECF in EMosis-CC/TERT1 cells; therefore, exposure to ECF induced strong oxidative stress. Development of a therapeutic strategy to reduce ROS production might be useful for preventing malignant transformation of endometriotic cysts.     

Application of Streptomyces pactum Act12 Enhances Drought Resistance in Wheat

The use of beneficial microbes to improve drought resistance in crops has great application potential in agricultural production, yet the effects of actinomycetes upon crop resistance to drought are rarely reported. Streptomyces pactum Act12 is a known multi-functional biocontrol agent of soil-borne diseases in several horticultural crops and medicinal plants. Here, we systematically analyzed how Act12 treatment affects drought resistance in drought-sensitive wheat (Triticum aestivum L.) cultivar Xinong 979 by considering both its effects and underlying mechanisms. After seed exposure to a cell-free culture filtrate of Act12, we measured several plant growth variables, osmotic adjustment and antioxidant capacity, cell membrane peroxidation, and drought resistance-related gene expression in wheat seedlings under drought stress conditions simulated by polyethylene glycol 6000. Results showed that, under drought stress, wheat seedling exposure to Act12 cell-free filtrate facilitated plant growth, with significant increases in shoot fresh weight (21.3%), shoot length (10.3%), and root length (13.6%). Act12 treatment also significantly increased total soluble sugar content in wheat leaves while decreasing their malondialdehyde content by 20.5%. Under non-drought conditions, Act12 treatment increased the content of both proline and glutathione in wheat leaves; however, both were lowered in Act12-treated plants compared with non-treated plants at 96 h of drought stress. Further analysis revealed that Act12 treatment increased the content of leaf abscisic acid and upregulated the expression levels of several drought resistance-related genes, such as EXPA2, EXPA6, P5CS, and SnRK2. These results suggest that application of S. pactum Act12 can enhance the osmotic adjustment and antioxidant capacity of plants via induction of abscisic acid accumulation and up-regulation of drought resistance-related gene expression, thereby mitigating drought stress impact in wheat.

     

Effect of <Emphasis Type="Italic">Medicago sativa</Emphasis> ferritin gene on stress tolerance in transgenic grapevine

The effect of Medicago sativa (alfalfa) ferritin gene (MsFer) on abiotic stress tolerance was tested using transgenic Vitis berlandieri × Vitis rupestris cv. ‘Richter 110’ grapevine rootstock lines. Leaf discs from transgenic plants maintained higher photosynthetic activity after NaCl, tert-butyl-hydroperoxide (t-BHP) or paraquat treatment than control ones. These results indicate that the increased production of ferritin significantly improved abiotic stress tolerance in transgenic grapevine plants.     

Zinc Uptake Regulator (<Emphasis Type="Italic">zur</Emphasis>) Gene Involved in Zinc Homeostasis and Virulence of <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> in Rice

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, one of the most widespread and destructive bacterial diseases in rice. In order to understand the gene of zinc uptake regulator (zur) involved in virulence of the pathogen in rice, we generated a mutant OSZRM by homologous suicide plasmid integration. The mutant failed to grow in NYGB medium supplemented with Zn²⁺ or Fe³⁺ at a concentration of 500 μM or 6 mM, whereas the wild-type strain grew well at the same conditions. The zur mutant was hypersensitive to hydrogen peroxide and exhibited reduction catalase activity and the production of extracellular polysaccharide (EPS). Interestingly, the mutant showed a reduction in virulence on rice but still kept triggering hypersensitive response (HR) in tobacco. When the mutant was complemented with the zur gene, the response was recovered to wild-type. These results suggested that zur gene is a functional member of the Zur regulator family that controls zinc and iron homeostasis, oxidative stress, and EPS production, which is necessary for virulence in X. oryzae pv. oryzae. Wanfeng Yang and Yan Liu contributed equally to this work     

Alleviating the inhibitory effect of salinity stress on nod gene expression in Rhizobium tibeticum – fenugreek (Trigonella foenum graecum) symbiosis by isoflavonoids treatment

Rhizobia-legume symbiosis depends on molecular dialog, which involves the production of specific plant flavonoid compounds as signal molecules. Rhizobium tibeticum was recovered from the root nodule of fenugreek and identified by sequencing the 16S rRNA gene. The effect of salinity stress on nod gene expression was measured in terms of β-galactosidase activity. R. tibeticum containing Escherichia coli lacZ gene fusions to specific nodulation (nod) genes were used to determine β-galactosidase activity. Combination of hesperetin (7.5 µM) and apigenin (7.5 µM) significantly increased β-galactosidase activity more than the single application of hesperetin or apigenin. Preincubation of R. tibeticum with hesperetin and apigenin combination significantly alleviates the adverse effect of salinity on nod gene expression and therefore, enhances nodulation and nitrogen fixation of fenugreek.     

Diversity of <Emphasis Type="Italic">nifH</Emphasis> gene amplified from plankton-community DNA in a shallow eutrophic lake (Lake Donghu,Wuhan, China)

Biological nitrogen fixation (BNF) is one of the major nitrogen inputs into the biosphere, and the nitrogenase iron protein (nifH) gene plays important roles in regulating the molecular nitrogen (N₂) fixation process. The nifH gene has also been extensively used to study the diversity and function of nitrogen-fixing microorganisms. In this study, we investigated the diversity of the nifH gene by culture-independent methods to analysis the planktonic nitrogen-fixing organisms in Lake Donghu, Wuhan, the largest urban lake in China. Results indicate that nifH gene sequences cloned from planktonic-community DNA showed high similarity to the uncultured cyanobacterial sequences deposited in the GenBank database. Phylogenetic analysis on the basis of the translated amino acid sequences further showed that most nifH clones were closely related to the reported cyanobacterial nifH gene sequences. Results also indicate that there are similar planktonic nitrogen-fixing organisms in the relatively independent areas of Lake Donghu, even though different regions showed a wide gradient in trophic status. These and other observations led us to believe that studies on nifH gene diversity and expression will increase our ability to understand the ecological function of target nitrogen-fixing groups in aquatic ecosystems.     

Nitrogenase Inhibition in Nodules from Pea Plants Grown Under Salt Stress Occurs at the Physiological Level and can be Alleviated by B and Ca

In order to shed new light on the mechanisms of salt-mediated symbiotic N₂-fixation inhibition, the effect of salt stress (75 mM) on N₂-fixation in pea root nodules induced by R. leguminosarum was studied at the gene expression, protein production and enzymatic activity levels. Acetylene reduction assays for nitrogenase activity showed no activity in salt-stressed plants. To know whether salt inhibits N₂-fixing activity at a molecular or at a physiological level, expression of the nifH gene, encoding the nitrogenase reductase component of the nitrogenase enzyme was analyzed by RT-PCR analysis of total RNA extracted from nodulated roots. The nifH messenger RNA was present both in plants grown in the presence and absence of salt, although a reduction was observed in salt-stressed plants. Similar results were obtained for the immunodetection of the nitrogenase reductase protein in Western-blot assays, indicating that nitrogen fixation failed mainly at physiological level. Given that nutrient imbalance is a typical effect of salt stress in plants and that Fe is a prosthetic component of nitrogenase reductase and other proteins required by symbiotic N₂-fixation, as leghemoglobin, plants were analyzed for Fe contents by atomic absorption and the results confirmed that Fe levels were severely reduced in nodules developed in salt-stressed plants. In a previous papers (El-Hamdaoui et al., 2003b), we have shown that supplementing inoculated legumes with boron (B) and calcium (Ca) prevents nitrogen fixation decline under saline conditions stress. Analysis of salt-stressed nodules fed with extra B and Ca indicated that Fe content and nitrogenase activity was similar to that of non-stressed plants. These results indicate a linkage between Fe deprivation and salt-mediated failure of nitrogen fixation, which is prevented by B and Ca leading to increase of salt tolerance.