The aadE*-sat4-aphA-3 Gene Cluster of Mycoplasma bovirhinis HAZ141_2 Undergoes Genomic Rearrangements Influencing the Primary Promoter Sequence

The 54 kb GC-rich prophage region of Mycoplasma bovirhinis HAZ141_2 contains three structural ‘compartments’, one of which is a highly transmittable cluster of three genes, aadE-like (aadE*), sat4, and aphA-3. In this study, we characterized recombination events and their consequences occurred within the aadE*-sat4-aphA-3 containing region. Analysis of this region revealed direct repeats (DRs) of 155 and invert repeats (IRs) of 197 base pairs (bps) each, flanking and overlapping with the primary promoter P* located upstream of the aadE*. Two recombination events, including inversions via both 197 and 155-bp IRs (the latter become inverted after the initial 197-bp IRs associated inversion) and the excision of the aadE*-sat4-aphA-3 cluster, were confirmed. Inversion via 155-IRs results in changes within the P* promoter region. Using Escherichia coli JM109 carrying plasmids containing derivatives of the aadE*-sat4-aphA-3 cluster, we validated the expression of those genes from different promoters. Our results showed no difference in the minimal inhibitory concentrations (MICs) to kanamycin and neomycin and only 2-fold decrease in MIC (from 512 to 256 μg/mL) to nourseothricin between the wild type and a P* derivative promoter. However, the MICs to kanamycin and neomycin were at least 4-fold lower in the construct where aphA-3 expressed under its P2 promoter (128 µg/mL) in comparison to the construct where aphA-3 expressed under P1” promoter located within the sat4 gene (512–1024 µg/mL). PCR confirmed the excision of the aadE*-sat4-aphA-3 cluster via 197- and 155-bp DRs, but no selection of antibiotic-sensitive M. bovirhinis were obtained after 100 passages in kanamycin-free medium.

The molecular characterization of the coat protein sequence and differentiation of CMV- subgroup I on tobacco from native flora in Turkey

Cucumber mosaic virus (CMV) has a broad plant-host range and a wide ecological zone distribution. Virus-like symptoms were observed on tobacco fields of Adiyaman province (Turkey) showing conspicuous mottling, greenish mosaic patterns and severe malformations of leaves. A total of forty tobacco samples tested positive against CMV by reverse transcription polymerase chain reaction (RT-PCR) using coat protein gene specific primers. Five randomly chosen CMV isolates were cloned into pGEM T-Easy vector and transformed into Escherichia coli JM109 strain. The recombinant bacterial clones containing insert-DNA were further purified and sequenced bidirectionally. In multiplex-RT-PCR studies carried out, it was found that all 40 CMV isolates belong to Subgroup I by resulting a 593 bp long DNA fragments. CMV subgroup IA was found to predominate in 4 out of 5 tobacco samples and CMV subgroup IB was found in 1 out of 5 CMV-positive samples by comparing the isolates with CMV reference isolates in phylogenetic tree. However, no Subgroup II sequences were found by multiplex RT-PCR using discriminating primers. The nucleic acid sequences were analyzed for the investigation of diversity of coat protein (CP) sequences of 5 CMV isolates. The sequence similarity ranged from 94.2-100% with the CMV subgroup I isolates infecting diverse plants in other regions of the world. The evolutionary tree revealed that the CMV IA Adiyaman isolates exhibited a genetic affinity with Australian and Spanish isolates. However, the CMV IB Adiyaman isolate showed a close genetic relationship with only the Australian isolates. To our knowledge, this study shows for the first time the occurrence of CMV IA and IB isolates infecting cultured tobacco plants in Adiyaman province.

KLONING GEN carB Salmonella typhi MENGGUNAKAN VEKTOR EKSPRESI pET-16b dan SEL INANG Escherichia coli JM109

The effort of cloning the Lister strain of Salmonella typhi (NCTC 786, BCC 712) carB gene using pET-16b expression vector and E. coli JM109 as host cell has been done. The carB gene and the pET-16b expression vector were both prepared from their recombinant plasmid digested using BamHI and NdeI as restriction enzymes. The pG-carB-11-ST recombinant plasmid was isolated from Escherichia coli XL10(pG-carB-11-ST) and the pET-carA-ST recombinant plasmid was from Escherichia coli DH5α(pET-carA-ST). After being ligated (in a ratio of vector:gene of 1:3, 1:5 and 1:6) in the presence of T4 Bacteriofage DNA Ligase, the ligation mixture was used to transform Escherichia coli JM109 cells and plated out onto Luria Bertani medium containing ampicillin. An amount of 369 produced colonies were screened for the presence of the appropriate recombinant plasmid using combination of plasmid miniprep and agarose gel electrophoresis. None of the recombinant plasmids being suspected to carry the Salmonella typhi carB gene. It is suggested to repeat cloning process using E. coli JM109 or using E. coli XL10 as host cell which were known to have large cloning efficiency and can be used for large plasmids up to 25 kb.

Study on Transglucosylation Properties of Amylosucrase from Xanthomonas campestris pv. Campestris and Its Application in the Production of α-Arbutin

α-Arbutin (4-hydroquinone-α-D-glucopyranoside), an effective skin-lightening agent due to its considerable inhibitory effect on human tyrosinase activity, is widely used in the pharmaceutical and cosmetic industries. Recently, α-arbutin was prepared through transglucosylation of hydroquinone using microbial glycosyltransferases as catalysts. However, the low yield and prolonged reaction time of the biotransformation process of α-arbutin production limited its industrial application. In this work, an amylosucrase (ASase) from Xanthomonas campestris pv. campestris str. ATCC 33913 (XcAS) was expressed efficiently in Escherichia coli JM109. The catalytic property of the purified XcAS for the synthesis of α-arbutin was tested. The recombinant strain was applied for highly efficient synthesis of α-arbutin using sucrose and hydroquinone as glucosyl donor and acceptor, respectively. By optimizing the biotransformation conditions and applying a fed-batch strategy, the final production yield and conversion rate of α-arbutin reached 60.9 g/L and 95.5%, respectively, which is the highest reported yield by engineered strains. Compared to the highest reported value (<1.4 g/L/h), our productivity (7.6 g/L/h) was improved more than five-fold. This work represents an efficient and rapid method for α-arbutin production with potential industrial applications.

Antibacterial Mechanism of Gloverin2 from Silkworm, Bombyx mori

Gloverin is one of the glycine-rich antimicrobial peptide exclusively found in Lepidoptera insects. It is generally activated through the innate immune system in insects. In this study, recombinant Gloverin2 from Bombyx mori (BmGlv2) was synthesized using a prokaryotic expression system. Circular dichroism spectroscopy showed that the recombinant BmGlv2 has random coil structure, which is relatively stable at the temperatures ranging from 15 to 82.5 °C. Antimicrobial activity analysis revealed that BmGlv2 significantly inhibited the growth of gram-negative bacteria, Escherichia coli JM109 and Pseudomonas putida, by disrupting cell integrity. Western blotting and immunofluorescence analyses suggested that BmGlv2 absorbed on the cell surface after incubation, which might be the first step in the antibacterial process. Our results also proved that the cell wall component lipopolysaccharides (LPS) induce a conformational change in BmGlv2 from a random coil to α-helix. Subsequently, α-helical BmGlv2 would recruit more BmGlv2 and form higher aggregation state. Collectively, these findings expand our understanding of antibacterial mechanism of BmGlv2.

Cloning and expression of recombinant thrombin in Escherichia coli JM109 (DE3)

Prothrombin, a protein involved in blood coagulation, is a plasma glycoprotein composed of the Gla domain, two adjacent kringle domains, and a serine protease domain. Prothrombin is a thrombin precursor playing the important role in the coagulation physiological as well as pathological condition. Thrombin is the key to convert the fibrinogen into fibrin by switching activation of XIII factor, pushed plasminogen into plasmin, the develope of the fibroblast and helps the stabilization of thrombolysis. In this study, the prothrombin gene was 936 bp in lengths and encoded 312 amino acids from bovine lung was optimized codon, was cloned in pET21a+ vector and expression in E. coli, in order to replace traditional bandages having slow affect, reduce the cost of products, cater the community health. The results showed that initially the successful cloning and expression of recombinant prothrombin in E. coli JM109 (DE3). Prothrombin, 1 glycoprotein huyết tương liên quan tới quá trình đông máu gồm 2 vùng Gla, 2 vùng Kringle và 1 vùng serine protease. Prothrombin là tiền chất của thrombin có vai trò quan trọng trong sinh lý đông máu cũng như tình trạng bệnh lý. Thrombin được xem như chìa khóa để chuyển hóa fibrinogen thành fibrin bằng cách hoạt hóa các yếu tố đông máu như XIII, thúc đẩy chuyển plasminogen thành plasmin và kích thích tăng sinh các tế bào tơ (fibroblast), giúp ổn định quá trình làm tan huyết khối. Trong nghiên cứu, các gen prothrombin được tách dòng từ phổi bỏ có kích thước 936 bp, mã hóa cho 312 axit amin được tối ưu hóa codon, nhân dòng vào vector pET21a+ và biểu hiện trong E. coli. Mục đích của nghiên cứu nhằm tạo ra băng gạc cầm máu nhanh, giá thành rẻ, phục vụ sức khỏe cộng đồng và thay thể băng gạc truyền thống. Kết quả nghiên cứu bước đầu cho thấy đã nhân dòng và biểu hiện thành công prothrombin tái tổ hợp ở chủng E. coli JM109 (DE3).