Somatic homologous recombination in planta: The recombination frequency is dependent on the allelic state of recombining sequences and may be influenced by genomic position effects

The Escherichia coli sodA gene encoding the antioxidant enzyme Mn-containing superoxide dismutase (MnSOD), was cloned in the expression vector pMG36e. This vector has a multiple cloning site down-stream of a promoter and Shine-Dalgarno sequences derived from Lactococcus. The protein-coding region of sodA from E. coli was amplified by the polymerase chain reaction, using a thermocycler and Taq DNA polymerase before cloning into pMG36e. When introduced into E. coli, the recombinant plasmid expressed the predicted fusion protein, both in the presence and absence of oxygen. The expression of the fusion protein in E. coli was verified by SOD assays, activity gels and Western blots. The recombinant plasmid was also introduced into Lactococcus lactis, which contains a resident SOD, and into Lactobacillus gasseri, which is devoid of SOD. Transformed lactococci expressed an active SodA fusion protein plus an active hybrid protein composed of subunits of the Lactococcus and the recombinant E. coli enzymes. Transformants of L. gasseri expressed only the fusion SodA protein, which was enzymatically active.     

Design,expression and characterization of recombinant hybrid peptide Attacin-Thanatin in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Antimicrobial peptides will be attractive and potential candidates as peptide drugs because of their efficient action against microbes and low toxicity to mammal cells. To improve their antibacterial activity, some modifications needs to be made. In this research, the hybrid peptide gene Attacin-Thanatin with 642 bp in length with preferred codons of E. coli was generated using the technology of Gene splicing by overlap extension. The gene was inserted in-frame into E. coli expression plasmid pET-32a (+) and induced to express in E. coli Rosetta. The recombinant protein was partial purified and its biological activity was determined. Analysis of the E. coli Rosetta induced with IPTG revealed that the molecular weight of fusion protein was approximately 41.8 kDa, which perfectly matched the mass calculated from the amino acid sequence. Biological activity detection showed that this peptide effectively inhibited the growth of the test bacteria including E. coli DH5α, E. coli BL21 (DE3), Salmonella choleraesuis and Staphylococcus aureus. Among these bacteria, the Gram-negative E. coli was the most sensitive. Furthermore, there was minor hemolysis activity for porcine red blood cells. So, the results indicated that the hybrid peptide Attacin-Thanatin could be served as a promising candidate for the chemical antibiotics.     

Antimicrobial cationic surfactant, cetyltrimethylammonium bromide, induces superoxide stress in Escherichia coli cells

Aims: To clarify whether an antibacterial surfactant, cetyltrimethylammonium bromide (CTAB), induces superoxide stress in bacteria, we investigated the generation of superoxide and hydrogen peroxide and expression of soxR, soxS and soxRS regulon genes in Escherichia coli cells with the treatment of CTAB. Methods and Results: In situ oxidative stress analyses with BES fluorescent probes revealed that generation of both superoxide and hydrogen peroxide were significantly increased with the CTAB treatment at a sublethal concentration in wild‐type strain OW6, compared with the CTAB‐resistant strain OW66. The activity of manganese–superoxide dismutase (Mn–SOD), a member of the soxRS regulon proteins, was decreased by the CTAB treatment only in strain OW6. Furthermore, quantitative real‐time PCR analyses revealed that expression of the soxRS regulon genes was not upregulated, although soxS was upregulated by the CTAB treatment in strain OW6. Conclusions: Cetyltrimethylammonium bromide treatment led E. coli cells to a generation state of superoxide and hydrogen peroxide. It was also suggested that superoxide generation was caused by inhibiting SoxS function and decreasing Mn–SOD activity. Significance and Impact of the Study: It was revealed that excess superoxide generation in bacterial cells play a key action of antibacterial surfactants.     

Molecular cloning,expression in <Emphasis Type="Italic">Escherichia</Emphasis><Emphasis Type="Italic">coli</Emphasis> of Attacin A gene from <Emphasis Type="Italic">Drosophila</Emphasis> and detection of biological activity

Attacin, a 20 kDa antibacterial peptide, plays an important role in immunity. To understand this gene better, gene cloning, expression and biological activity detection of Attacin A was carried out in present study. The full-length open reading frame (ORF) coding for Attacin A gene was generated using RT-PCR which takes total RNA extracted from Drosophila as the template. The gene was inserted directionally into the prokaryotic expression vector pET-32a (+). The resulting recombinant plasmid was transformed into E. coli Rosetta. SDS–PAGE was carried out to detect the expression product which was induced by IPTG. The antimicrobial activity and hemolysis activity were tested in vitro after purification. Agarose gel electrophoresis indicated that the complete ORF of Attacin A gene has been cloned successfully from Drosophila stimulated by E. coli which includes 666 bp and encodes 221 AA. The gene encoding mature Attacin A protein was amplified by PCR from the recombinant plasmid containing Attacin A, which includes 570 bp in all. SDS–PAGE analysis demonstrated that the fusion protein expressed was approximately 39.2 kDa. Biological activities detection showed that this peptide exhibited certain antibacterial activity to several G− bacteria, as well as minor hemolysis activity for porcine red blood cells. In conclusion, Attacin A gene was cloned and expressed successfully. It was the basis for further study of Attacin.     

Production and purification of a novel antibiotic peptide,adenoregulin, from a recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Adenoregulin is a member of dermaseptin family which are vertebrate antibiotic peptides having lethal effects against a broad spectrum of bacteria, fungi and protozoa. The 99 bp adenoregulin gene was cloned in the expression vector pET32a and transformed into Escherichia coli BL21(DE3). In fed-batch cultivation of BL21(DE3)/pET32a-adr, an exponential feeding strategy was applied to gain 60 g dry cells l^–1. The recombinant fusion protein Trx-ADR was expressed in a soluble form. The fusion protein was isolated by Ni²⁺-chelating chromatography, cleaved with CNBr and purified to homogeneity through reverse phase-HPLC and size exclusion-HPLC. The purified recombinant adenoregulin had antibacterial activity against Escherichia coli K12D31 with apparent Mr of 3.4 kDa, identical to the anticipated value.     

Somatic homologous recombination in planta: The recombination frequency is dependent on the allelic state of recombining sequences and may be influenced by genomic position effects

The Escherichia coli sodA gene encoding the antioxidant enzyme Mn-containing superoxide dismutase (MnSOD), was cloned in the expression vector pMG36e. This vector has a multiple cloning site down-stream of a promoter and Shine-Dalgarno sequences derived from Lactococcus. The protein-coding region of sodA from E. coli was amplified by the polymerase chain reaction, using a thermocycler and Taq DNA polymerase before cloning into pMG36e. When introduced into E. coli, the recombinant plasmid expressed the predicted fusion protein, both in the presence and absence of oxygen. The expression of the fusion protein in E. coli was verified by SOD assays, activity gels and Western blots. The recombinant plasmid was also introduced into Lactococcus lactis, which contains a resident SOD, and into Lactobacillus gasseri, which is devoid of SOD. Transformed lactococci expressed an active SodA fusion protein plus an active hybrid protein composed of subunits of the Lactococcus and the recombinant E. coli enzymes. Transformants of L. gasseri expressed only the fusion SodA protein, which was enzymatically active.     

Immune function of a Rab‐related protein by modulating the JAK‐STAT signaling pathway in the silkworm,Bombyx mori

The Rab‐family GTPases mainly regulate intracellular vesicle transport, and play important roles in the innate immune response in invertebrates. However, the function and signal transduction of Rab proteins in immune reactions remain unclear in silkworms. In this study, we analyzed a Rab‐related protein of silkworm Bombyx mori (BmRABRP) by raising antibodies against its bacterially expressed recombinant form. Tissue distribution analysis showed that BmRABRP mRNA and protein were high expressed in the Malpighian tubule and fat body, respectively. However, among the different stages, only the fourth instar larvae and pupae showed significant BmRABRP levels. After challenge with four pathogenic microorganisms (Escherichia coli, BmNPV, Beauveria bassiana, Micrococcus luteus), the expression of BmRABRP mRNA in the fat body was significantly upregulated. In contrast, the BmRABRP protein was significantly upregulated after infection with BmNPV, while it was downregulated by E. coli, B. bassiana, and M. luteus. A specific dsRNA was used to explore the immune function and relationship between BmRABRP and the JAK‐STAT signaling pathway. After BmRABRP gene interference, significant reduction in the number of nodules and increased mortality suggested that BmRABRP plays an important role in silkworm's response to bacterial challenge. In addition, four key genes (BmHOP, BmSTAT, BmSOCS2, and BmSOCS6) of the JAK‐STAT signaling pathway showed significantly altered expressions after BmRABRP silencing. BmHOP and BmSOCS6 expressions were significantly decreased, while BmSTAT and BmSOCS2 were significantly upregulated. Our results suggested that BmRABRP is involved in the innate immune response against pathogenic microorganisms through the JAK‐STAT signaling pathway in silkworm.     

Senescence‐associated superoxide dismutase influences mitochondrial gene expression in budding tunicates

A recent study has shown that in the budding tunicate Polyandrocarpa misakiensis, the mitochondrial respiratory chain (MRC) dramatically attenuates the gene activity during senescence. In this study, we examined the possible involvement of superoxide dismutase (SOD) in the attenuation of gene expression of cytochrome c oxidase subunit 1 (COX1) in aged zooids. By RT‐PCR and in situ hybridization, Cu/Zn‐SOD (SOD1) was found to be expressed in most cells and tissues of buds and juvenile zooids but showed a conspicuous decline in senescent adult zooids, except in the gonad tissue in which the cytoplasm of juvenile oocytes was stained heavily. This expression pattern of SOD1 was similar to that of COX1. In contrast to SOD1, Mn‐SOD (SOD2) was expressed constitutively in both somatic and germline tissues of buds, juvenile zooids, and senescent adult zooids. Knockdown of SOD1 by RNAi diminished the gene activity of not only SOD1 but also of COX1. The resultant zooids had transient deficiencies in growth and budding, and they recovered from these deficiencies approximately 1 month later. Our results indicate that in P. misakiensis, SOD1 is a senescence‐associated nuclear gene and that the experimental decline in SOD1 gene expression accompanies the attenuation of MRC gene activity. Although it is uncertain how SOD1 is downregulated during tunicate senescence, the decreased SOD1 activity could be one of the main causes of MRC gene attenuation during normal senescence.     

Zinc deficiency tolerance in maize is associated with the up‐regulation of Zn transporter genes and antioxidant activities

• Zinc (Zn) is an essential micronutrient for the growth and development of plants. However, Zn deficiency is a common abiotic stress causing yield loss in crop plants. This study elucidates the mechanisms of Zn deficiency tolerance in maize through physiological and molecular techniques.
• Maize lines tolerant (PAC) and sensitive (DAC) to Zn deficiency were examined physiologically and by atomic absorption spectrometry (AAS). Proteins, H₂O₂, SOD, POD, membrane permeability and gene expression (using real‐time PCR) of roots and shoots of both maize lines were assessed.
• Zn deficiency had no significant effect on root parameters compared with control plants in PAC and DAC but showed a substantial reduction in shoot parameters in DAC. AAS showed a significant decrease in Zn concentrations in both roots and shoots of DAC but not PAC under Zn deficiency, implying that Zn deficiency tolerance mechanisms exist in PAC. Consistently, total protein and membrane permeability were significantly reduced in DAC but not PAC in both roots and shoots under Zn deficiency in comparison with Zn‐sufficient plants. Real‐time PCR showed that expression of ZmZIP1, ZmZIP4 and ZmIRT1 transporter genes significantly increased in roots of PAC, but not in DAC due to Zn deficiency compared with controls. The H₂O₂ concentration dramatically increased in roots of DAC but not PAC. Moreover, tolerant PAC showed a significant increase in POD and SOD activity due to Zn deficiency, suggesting that POD‐ and SOD‐mediated antioxidant defence might provide tolerance, at least in part, under Zn deficiency in PAC.
• This study provides an essential background for improving Zn biofortification of maize.

     

Aluminum toxicity related to SOD and expression of presenilin and CREB in Bombyx mori

Aluminum (Al) is an important environmental metal factor that can be potentially associated with pathological changes leading to neurotoxicity. The silkworm, Bombyx mori, is an important economic insect and has also been used as a model organism in various research areas. However, the toxicity of Al on silkworm physiology has not been reported. Here, we comprehensively investigate the toxic effects of Al on the silkworm, focusing on its effects on viability and development, superoxide dismutase (SOD) activity, and the expression of presenilin and cAMP response element‐binding protein (CREB) in BmE cells and silkworm larvae. BmE cell viability decreased after treatment with aluminum chloride (AlCl₃) in both dose‐ and time‐dependent manners. When AlCl₃ solution was injected into newly hatched fifth instar larvae, both larval weight gain and survival rate were significantly decreased in a manner correlating with AlCl₃ dose and developmental stage. Furthermore, when BmE cells and silkworm larvae were exposed to AlCl₃, SOD activity decreased significantly relative to the control group, whereas presenilin expression increased more than twofold. Additionally, CREB and phosphorylated CREB (p‐CREB) expression in the heads of fifth instar larvae decreased by 28.0% and 50.0%, respectively. These results indicate that Al inhibits the growth and development of silkworms in vitro and in vivo, altering SOD activity and the expressions of presenilin, CREB, and p‐CREB. Our data suggest that B. mori can serve as a model animal for studying Al‐induced neurotoxicity or neurodegeneration.     

Proteomic analysis of the immune response of the silkworm infected by Escherichia coli and Bacillus bombyseptieus

Abstract The silkworm, Bombyx mori, is an economically important insect with a 5 000‐year history of domestication. During evolution, the silkworm has developed highly effective defenses against invasion and parasitization by microorganisms. In this study, two microorganisms Escherichia coli and Bacillus bombyseptieus were orally infected to silkworm larvae. After infection with E. coli and B. bombyseptieus for 24 h, we investigated the polypeptide changes in the hemolymph, midgut and integument using two‐dimensional gel electrophoresis and matrix‐assisted laser desorption ionization time of flight mass spectrometry. Forty‐seven differentially expressed proteins were identified in these tissues. They belonged to a variety of functional classes, including immune proteins, metabolic proteins and structural proteins. Compared with controls, E. coli‐infected silkworms showed 21 up‐regulated proteins, 25 down‐regulated proteins and lost one protein. After infection with B. bombyseptieus, silkworms showed 15 up‐regulated proteins, 27 down‐regulated proteins, lost three proteins and retained two proteins unchanged. We speculate that all these proteins may play a role in the silkworm immune response, although it is unclear why and how the two kinds of bacteria can so markedly alter expression of these proteins. These results offer valuable insights for measuring the proteomic responses of the silkworm innate immune mechanism.     

High-level production of bioactive human beta-defensin-4 in Escherichia coli by soluble fusion expression

Human beta-defensin-4 (hBD4) is a cationic 50-amino acid antimicrobial peptide with three conserved cysteine disulfide bonds. It exhibits a broad antimicrobial spectrum. This study describes the synthesis of hBD4 gene, the heterologous fusion expression of the peptide in Escherichia coli, and the bioactive assay of released hBD4. A PCR-based gene SOEing (splicing by overlap extension) synthesis method was used in the synthesis of the hBD4 gene with optimized codons. By constructing the expression plasmid (pET32-smhBD4), high concentration of soluble hBD4 fusion protein (1.9 g/l) can be obtained in E. coli. Further optimization studies showed that the expression system was very efficient to produce soluble target protein, and the solubility of the target protein could attain more than 99% even when the culture temperature was as high as 37°C. The highest productivity (2.68 g/l) of the hBD4 fusion protein was achieved by cultivating the E. coli (pET32-smhBD4) in MBL medium at 34°C, inducing the culture at the mid-exponential phase with 0.4-mM isopropyl β-d-galactopyranoside (IPTG), and collecting the broth after 6-h expression. The soluble target protein accounted for 64.6% of the total soluble proteins, and the mature hBD4 expression level was stoichiometrically estimated to be 0.689 g/l. This fusion protein was then purified and cleaved to get the mature hBD4 peptide that showed antimicrobial activity against E. coli and Pseudomonas aeruginosa.     

Bacterium‐Expressed dsRNA Downregulates Microsporidia Nosema bombycis Gene Expression

The microsporidia Nosema bombycis is the insect pathogen of pebrine disease severely destructive to sericulture production. Here, we describe the use of Escherichia coli HT115 strain (DE3) to express double‐strand RNAs targeting the gene encoding ADP/ATP protein in N. bombycis. The results showed that dsRNAs deferentially suppressed the gene expression during N. bombycis infection in the silkworm, and the effect waned gradually. Our results, for the first time, provide a tool to utilize the dsRNA expressed by recombinant E. coli to control the pebrine disease of the domestic silkworm.     

Molecular characterization and expression analysis of BmNOX in two strains of Bombyx mori with contrasting viral resistance phenotype

We recently documented the identification of a 26.5 kDa protein named BmNox in the gut fluid of Nistari strain of Bombyx mori, which possessed antiviral activity against BmNPV in vitro. In this report, we report the characterization of the full‐length gene encoding BmNOX and the levels of expression of this gene in select tissues of silkworm larvae from a BmNPV‐susceptible and a BmNPV‐resistant strain to the defense capability in Bombyx mori larvae challenged with BmNPV. We also evaluated the BmNox expression in various stages of larval life of a resistant and a susceptible strain of Bombyx mori selected from among a panel of strains of silkworm. Nistari, a multivoltine strain of silkworm, expressed BmNOX during all five larval stages, and were highly resistant to BmNPV infection. In sharp contrast, CSR₂, a bivoltine strain, showed weaker expression of BmNOX in the anterior midgut in larval life and was highly susceptible to BmNPV infection. BmNOX is a secretory protein with dual expression in gut fluid and mid gut tissue. BmNOX is expressed heavily in the posterior mid gut, with weaker expression in the fore‐ and mid‐gut regions. © 2010 Wiley Periodicals, Inc.     

Construction of a dual-tag system for gene expression, protein affinity purification and fusion protein processing

An E. coli vector system was constructed which allows the expression of fusion genes via a l-rhamnose-inducible promotor. The corresponding fusion proteins consist of the maltose-binding protein and a His-tag sequence for affinity purification, the Saccharomyces cerevisiae Smt3 protein for protein processing by proteolytic cleavage and the protein of interest. The Smt3 gene was codon-optimized for expression in E. coli. In a second rhamnose-inducible vector, the S. cerevisiae Ulp1 protease gene for processing Smt3 fusion proteins was fused in the same way to maltose-binding protein and His-tag sequence but without the Smt3 gene. The enhanced green fluorescent protein (eGFP) was used as reporter and protein of interest. Both fusion proteins (MalE-6xHis-Smt3-eGFP and MalE-6xHis-Ulp1) were efficiently produced in E. coli and separately purified by amylose resin. After proteolytic cleavage the products were applied to a Ni-NTA column to remove protease and tags. Pure eGFP protein was obtained in the flow-through of the column in a yield of around 35% of the crude cell extract.     

Molecular and genetic characterization of superoxide dismutase in Haemophilus influenzae type b

Oxygen free radicals present a serious potential threat to microbial survival, through their ability to inflict Indiscriminate damage on proteins and DNA. Superoxide dismutase (SOD, EC 1.15.1.1), among other oxygen-metabolizing enzymes, is essential to prevent these toxic molecules from accumulating in the bacterial cytosol during aerobic metabolism. The gene sodA, encoding manganese-containing SOD ([Mn]-SOD), has been cloned from a virulent strain of Haemophilus influenzae type b using degenerate oligonucleotides encoding regions of the gene conserved across different bacterial species. The gene product has been identified as [Mn]-SOD by its similarity at key amino acid residues to known examples of the enzyme, by expression of enzymatically active protein from cloned DNA expressed in Escherichia coli, and by demonstration that an in-frame deletion in the gene abolishes this activity. In contrast to the situation in E. coli, this [Mn]-SOD is the only active SOD detected in H. influenzae. In further contrast to E. coli, [Mn]-SOD gene expression in H. influenzae has been found to be only partially repressed under anaerobic conditions. When expressed in E. coli the gene is regulated by Fur and Fnr, and the promoter region, identified experimentally, has been found to contain nucleotide sequence motifs similar to the Fur- and Fnr-binding sequences of E. coli, suggesting the involvement of analogues of these aerobiosis- responsive activators in H. influenzae gene expression.     

Functional expression,purification, and antimicrobial activity of a novel antimicrobial peptide MLH in Escherichia coli

In this study, a novel heterozygous antimicrobial peptide MLH was synthesized, expressed, purified, and characterized. The peptide Md-cec-LL-37_Hp (MLH) was selected through bioinformatic analysis using musca domestica antimicrobial peptide (Cec-Med), human antimicrobial peptide LL-37, and helicobacter pylori antimicrobial peptide (Hp) as parent peptides. The target gene was synthesized by overlap extension PCR (SOE-PCR) and connected to the expression vector pET-32a (+), and the recombinant plasmid pET-32a-MLH was transformed to Escherichia coli for constructing pET-32a-MLH/BL21 (DE3). Isopropyl β-D-thiogalactoside (IPTG) was used to induce protein expression, and SDS-PAGE and western blot were adopted to test the target protein. And fermentation condition was optimized to get the mass expression of the fusion protein. The Ni²⁺ affinity chromatographic column was used to purify. Active heterozygous peptide was obtained after renaturation. Finally, the activity of the heterozygous antimicrobial peptide was identified. The fusion peptide showed significant antimicrobial effect on both E. coli and Staphylococcus aureus.