Multiplex PCR for simultaneous detection of enterococcal genes vanA and vanB and staphylococcal genes mecA, ileS-2 and femB

The experimental transfer of the vanA gene cluster from Enterococcus faecalis to Staphylococcus aureus has raised fears about the occurrence of such genetic transfer in clinical isolates of methicillin-resistant staphylococci. Recently, infections by a S. aureus strain carrying the enterococcal vancomycin resistance vanA gene cluster were reported. The possible emergence and dissemination of these strains is a serious health threat and makes optimization of prevention strategies and fast detection methods absolutely necessary. In the present study, we developed a PCR protocol for simultaneous detection of enterococcal vanA and vanB genes , the staphylococcal methicillin and mupirocin resistance markers mecA and ileS-2, and identification of S. aureus. As no vancomycin-resistant S. aureus isolates were available for our study, we used mixtures of enterococcal and staphylococcal colonies that harbored the different resistance markers to show that these genes could be detected simultaneously. This protocol could be used to facilitate the detection and identification of predictable S. aureus or methicillin-resistant strains carrying vanA or vanB.     

Overproduction of d-xylose isomerase in Escherichia coli by cloning the d-xylose isomerase gene

The Escherichia coli d-xylose isomerase (d-xylose ketol-isomerase, EC 5.3.1.5) gene, xylA, has been cloned on various E. coli plasmids. However, it has been found that high levels of overproduction of the d-xylose isomerase, the protein product of the xylA gene, cannot be accomplished by cloning the intact gene on high copy-number plasmids alone. This is believed to be due to the fact that the expression of the gene through its natural promoter is highly regulated in E. coli. In order to overcome this, the xylA structural gene has been fused with other strong promoters such as tac and lac, resulting in the construction of a number of fused genes. Analysis of the E. coli transformants containing the fused genes, cloned on high copy-number plasmids, indicated that a 20-fold overproduction of the enzyme can now be obtained. It is expected that overproduction of the enzyme in E. coli can still be substantially improved through additional manipulation with recombinant DNA techniques.     

A Clostridium perfringens Vector for the Selection of Promoters

A promoter selection vector for Clostridium perfringens genes was constructed from a C. perfringens-Escherichia coli shuttle vector, pJIR418. The plasmid carries a promoterless chloramphenicol acetyltransferase gene (catP), derived from pIP401, downstream of the multiple cloning sites of pUC18. When a promoter region of the phospholipase C gene was inserted into one of the cloning sites, derivatives of C. perfringens strain 13 carrying the resultant plasmid acquired resistance to chloramphenicol. This plasmid should be a useful reporter system for C. perfringens genes.     

Cloning of heterologous genes specifying detrimental proteins on pUC-derived plasmids inEscherichia coli

A system is described that enables the cloning of genes specifying detrimental proteins inEscherichia coli. The system is based on pUC plasmids and was developed for the expression of theBacillus subtilis csaA gene, which is lethal when expressed at high levels. Suppressor strains that tolerate the presence of plasmids for high-level expression ofcsaA were isolated, which contained small cryptic deletion variants of the parental plasmid in high copy numbers. The cryptic plasmids consisted mainly of the pUC replication functions and lacked thecsaA region and selectable markers. The co-resident, incompatible, cryptic plasmids enabled the maintenance of thecsaA plasmids by reducing their copy number 20-fold, which resulted in a concomitant 3- to 7-fold reduction in the expression of plasmid-encoded genes. Strains carrying these cryptic endogenous plasmids proved to be useful for the construction of pUC-based recombinant plasmids carrying other genes, such as theskc gene ofStreptococcus equisimilis, which cannot be cloned in high copy numbers inE. coli. Several strategies to reduce production levels of heterologous proteins specified by plasmids are compared.     

Organization of the lux genes of photobacterium phosphoreum

The lux genes from Photobacterium phosphoreum (NCMB844) have been cloned into Escherichia coli in a plasmid containing the T7-bacteriophage promoter. By specific expression in vivo under the T7 promoter, five structural genes (luxA-E) coding for the fatty acid reductase and luciferase polypeptides were identified as well as a new gene, designated as luxF, which codes for a 26kDa polypeptide. This new gene is located between luxB and luxE and thus disrupts the structural gene order of luxCDABE found in the Vibrio genus. The luxF gene and the protein it codes for have recently been identified in other Photobacterium species and so appears to be widely distributed within this genus. Nucleotide sequencing of the luxF gene has shown it to code for a protein homologous to the luciferase subunits, coded by the luxA and luxB genes. Although this gene is not necessary for light emission in all luminescent bacteria, it must play an essential role in the biochemistry, physiology, or ecology of the luminescent system in species of the Photobacterium genus.     

Visualization of bioluminescence as a marker of gene expression in rhizobium-infected soybean root nodules

The linked structural genes lux A and lux B, encoding bacterial luciferase of a marine bacterium Vibrio harveyi, were fused with the nitrogenase nifD promoter from Bradyrhizobium japonicum and with the P1 promoter of pBR322. Both fusions were integrated into the B. japonicum chromosome by site-specific recombination. Soybean roots infected with the two types of rhizobium transconjugants formed nitrogen-fixing nodules that produced bright blue-green light. Cells containing the P1 promoter/lux AB fusion resulted in continuously expressed bioluminescence in both free-living rhizobium and in nodule bacteriods. However, when under control of the nifD promoter, luciferase activity was found only in introgen-fixing nodules. Light emission from bacteroids allowed us to visualize and to photograph nodules expressing this marker gene fusion in vivo at various levels of resolution, including within single, living plant cells. Localization of host cells containing nitrogen-fixing bacteroids within nodule tissue was accomplished using low-light video microscopy aided by realtime image processing techniques developed specifically to enhance extreme low-level luminescent images.     

Analysis of a post-translational steroid induction system for GIGANTEA in Arabidopsis

Background  

To investigate the link between the flowering time gene GIGANTEA (GI) and downstream genes, an inducible GI system was developed in Arabidopsis thaliana L. Heynh. Transgenic Arabidopsis plant lines were generated with a steroid-inducible post-translational control system for GI. The gene expression construct consisted of the coding region of the GI protein fused to that of the ligand binding domain of the rat glucocorticoid receptor (GR). This fusion gene was expressed from the constitutive cauliflower mosaic virus 35S promoter and was introduced into plants carrying the gi-2 mutation. Application of the steroid dexamethasone (DEX) was expected to result in activation of the GI-GR protein and its relocation from the cytoplasm to the nucleus.     

Construction of a fused operon consisting of the recA and kan (Kanamycin resistance) genes and regulation of its expression by the lexA gene

Summary The kanamycin resistance gene (kan) of transposon Tn5 was cloned into a derivative of plasmid pBR322. A DNA fragment containing the promoter-operator region of the recA gene was inserted into the promoter region of the cloned kan gene to produce a fused operon, recA-kan. Plasmid pMCR685 carrying recA-kan expressed a low level of activity of the kan gene product (kanamycin phosphotransferase; KPT) in the wildtype cells of Escherichia coli, while the plasmid showed an increased level of the activity in the Spr^- mutant cells which produce the inactive lexA protein. The KPT activity in the wildtype cells harboring the plasmid increased 6-to 11-fold upon treatment of the cells with mitomycin C or nalidixic acid, both of which are known to induce synthesis of recA protein.Expression of the recA-kan operon fusion was remakably repressed by the lexA gene cloned into a plasmid carrying the operon fusion. Higher concentrations of mitomycin C were required for maximal induction of KPT activity in the cells harboring the resulting plasmid pMCR687. These results strongly suggest that the lexA gene product can by itself repress the recA gene, and that pMCR687 is a useful vector to clone genes whose expression is harmful to the host cell growth.     

The effects of promoter on transient expression in conifer cell lines

Summary Protoplasts from suspension cultures of somatic embryos of white spruce (Picea glauca Moench Voss) were electroporated with plasmids containing the chimeric genes for chloramphenicol acetyl transferase (CAT) or -glucuronidase (GUS), under control of one of three promoters. Transient CAT gene expression of approximately equal magnitude resulted when the CAT gene was fused to either the cauliflower mosaic virus (CaMV) 35S promoter or the nopaline synthase (NOS) promoter. When the CAT gene was fused to a tandem repeat CaMV 35S promoter (pPBI-363), CAT enzyme activity compared to NOS or 35S promoters increased up to eightfold (cell line WS-34), and were up to 100-fold greater than control (electroporated without plasmid). Comparatively, protoplasts of black spruce (Picea mariana Mill) and jack pine (Pinus banksiana Lamb.), electroporated with pPBI-363, produced increases in CAT activity compared to control of 90-fold and 70-fold, respectively. White spruce (WS-34) protoplasts were subsequently electroporated with the GUS gene fused to the tandem repeat CaMV 35S promoter. Comparatively, GUS enzyme activity increased up to tenfold compared to GUS fused to a CaMV 35S promoter. The results indicated that transient expression of the CAT and GUS genes was influenced by the type of promoter and cell line used, as well as by electroporation conditions.NRCC No. 30498     

代谢工程改造酿酒酵母提高法尼醇产量

【目的】法尼醇(FOH,C₁₅H₂₆O)是一种具有芳香气味的非环状倍半萜醇,被广泛应用于化妆品和医学药物的工业化生产,也可作为航空燃料的理想替代品。具有食品级安全性的酿酒酵母细胞能够合成内源性法尼醇,但其产量很低,无法满足工业生产的需要。因此,需要采用代谢工程手段,改造法尼醇合成途径,以有效提高法尼醇在酿酒酵母中的产量。【方法】以酿酒酵母工业菌株CEN.PK2-1D为底盘细胞,强化甲羟戊酸途径中关键酶的表达水平和弱化麦角固醇合成分支途径,以提高法尼醇合成所需的直接前体物质法尼基焦磷酸(FPP);并分别表达催化FPP合成法尼醇的五种内源磷酸酶和两种异源合酶,筛选能高效合成法尼醇的磷酸酶或合酶。【结果】通过在CEN.PK2-1D(法尼醇产量<0.1mg/L)中强化表达甲羟戊酸途径中截短形式的HMG-CoA还原酶(tHMGR1)和FPP合酶(ERG20),使法尼醇产量提高约50.8倍,达到5.08 mg/L;使用HXT1启动子替换鲨烯合酶编码基因ERG9启动子以下调其表达水平,使法尼醇产量进一步提升47.1倍,达到239.17 mg/L。在此...     

A Useful Cloning Vector for Bacillus subtilis

We have constructed plasmid pDN1050 a new small cloning vector for Bacillus subtilis . pDN1050 harbors the origin of replication of Staphylococcus aureus plasmid pUB110 and the chloramphenicol resistance gene of S. aureus plasmid pC194. The plasmid is segregationally and structurally stable. Plasmid pDN1370, a low copy number mutant of pDN1050 was isolated and shown to harbor a mutation in the repA gene of the replication protein.     

High intensity and blue light regulated expression of chimeric chalcone synthase genes in transgenic Arabidopsis thaliana plants

Summary To establish a genetic system for dissection of light-mediated signal transduction in plants, we analyzed the light wavelengths and promoter sequences responsible for the light-induced expression of the Arabidopsis thaliana chalcone synthase (CHS) promoter fused to the -glucuronidase (GUS) marker gene. Transgenic A. thaliana lines carrying 1975, 523, 186, and 17 by of the CHS promoter fused to the GUS gene were generated, and the expression of these chimeric genes was monitored in response to high intensity light in mature plants and to different wavelengths of light in seedlings. Fusion constructs containing 1975 and 523 by of CHS promoter sequence behaved identically to the endogenous CHS gene under all conditions. Expression of these constructs was induced specifically in response to high intensity white light and blue light. The response to blue light was seen in the presence of the P_fr form of phytochrome. Fusion constructs containing 186 by of promoter sequence showed reduced basal levels of expression and only weak stimulation by blue light but were induced significantly by high intensity white light. These analyses showed that the expression of the A. thaliana CHS gene is responsive to a specific blue light receptor and that sequences between — 523 and — 186 by are required for optimal basal and blue light-induced expression of this gene. The experiments lay the foundation for a simple genetic screen for light response mutants.     

银白色葡萄球菌的发现、分布与致病性

银白色葡萄球菌(Staphylococcus argenteus)是金黄色葡萄球菌(S. aureus)的近缘新种,于2015年正式被鉴定、命名。在此之前,银白色葡萄球菌一直被认为是金黄色葡萄球菌种内的一个远源支系。该物种绝大多数菌株分离自人体,基因组上含有大量与金黄色葡萄球菌同源的毒力基因,可导致与金黄色葡萄球菌类似的临床感染和食物中毒症状。因为与人类健康相关,银白色葡萄球菌受到了越来越多的关注,目前已在20多个国家被报道。但是由于出现较晚,与金黄色葡萄球菌难以区分,银白色葡萄球菌的全球分布状况、原始生境和传播过程尚不清楚。本文从发现过程、分布、致病性和鉴定方法等方面综述了银白色葡萄球菌的最新研究进展。     

2016-2017年宁夏地区牛源金黄色葡萄球菌的全基因组分析

【目的】了解宁夏地区奶牛乳腺炎金黄色葡萄球菌(Staphylococcus aureus,SA)代表菌株的基因组序列基本特征,进一步探究其耐药基因型、毒力及进化关系,为兽医临床防治提供理论依据。【方法】采用纸片法对97株金黄色葡萄球菌临床分离株进行抗菌药物敏感性试验,同时进行葡萄球菌蛋白A(Staphylococcus aureus protein A,spa)分型、多位点序列分型(multilocus sequence typing,MLST),根据分型结果选取16株代表菌株进行全基因组测序,并对获得的测序序列进行处理分析。【结果】药敏试验结果显示97株分离株对18种抗菌药物存在不同程度的耐药,其中9株耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus,MRSA)对青霉素、氨苄西林、苯唑西林、头孢噻呋、磺胺异噁唑、红霉素、庆大霉素和克林霉素等8种抗菌药物完全耐药,甲氧西林敏感金黄色葡萄球菌(methicillin-sensitive Staphylococcus aureus,MSSA)菌株对青霉素、氨苄西林、磺胺异噁唑耐...     

Co-ordinate expression of glycine betaine synthesis genes linked by the FMDV 2A region in a single open reading frame in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The genes encoding the two enzymes choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH) of glycine betaine synthesis in Suaeda salsa were cloned and fused with the 2A region of foot-and-mouth disease virus in a single open reading frame. The fused genes were placed under the control of the alcohol oxidase (AOX1) promoter in pPIC3B and transformed into P. pastoris GS115. The expression of the fused genes in P. pastoris and the ability of recombinant yeasts to tolerate environmental stresses were studied. The results showed that induced with 0.5% methanol for 96 h, the maximal activities of CMO and BADH in the tested recombinant yeasts were 45- and 44-fold higher than those in the control yeast transformed empty vector only, respectively; the content of glycine betaine in the recombinant yeasts was 28- to 35-fold higher than that in the control. The fused genes linked by 2A region of foot-and-mouth disease virus were expressed in P. pastoris successfully and the polyprotein was ‘cleaved’ to each functional protein. The yeasts transformed the fused genes, which were more resistant to salt, methanol, and high temperature stresses than the control as result of glycine betaine synthesis genes introduced.     

Tissue-dependent enhancement of transgene expression by introns of replacement histone H3 genes of Arabidopsis

Intron-bearing replacement histone H3 genes in Arabidopsis and other plants are highly and constitutively expressed. We demonstrate that the introns located within the 5-untranslated regions (5-UTR) of the two Arabidopsis replacement H3 genes will abolish the cell cycle dependence of an endogenous histone H4 promoter. We demonstrate that these introns, functionally combined with their endogenous promoters, could produce the high and constitutive expression of the replacement H3 genes observed in planta. They strongly increase gene expression whatever the promoter, from the strong 35S CaMV promoter to complete and resected promoters of cell cycle-dependent and replacement histone genes. Quantitative analysis of the extent of reporter gene enhancement in different parts of developing transgenic plantlets, ranging from 2-fold to 70-fold, supports the notion that trans-acting factors are responsible for this effect. Such factors appear most abundant in roots.