Gene Expression in Bacteria Directed by Plant-specific Regulatory Sequences

The regulation of gene expression represents a specific process which has different structural and functional requirements in different groups of organisms. It is thus assumed that regulatory sequences of eucaryotes cannot be recognized in procaryotes. This assumption is of interest for risk assessments of the environmental impact of deliberate release experiments with genetically modified organisms. In order to analyse the extent of heterologous gene expression caused by the transfer of plant-specific regulatory sequences into bacteria, we constructed fusions between plant-specific regulatory sequences and the coding regions of the luxAB genes for the luciferase of the bioluminescent bacterium Vibrio harveyi, transferred the fusions into different bacterial species and measured the luminescence to quantify the expression of the luciferase genes. The regulatory sequences investigated included (a) the 35S promoter of the Cauliflower mosaic virus, (b) the B33 promoter of a class I patatin gene of potatoes, (c) the promoter of the ST-LS1 gene of potatoes and (d) the promoter of the rolC gene of Agrobacterium rhizogenes. We could show that in addition to the 35S promoter, which has already been described as being recognized in Escherichia coli, the sequences containing the B33 and the ST-LS1 promoters are recognized in bacteria. Luciferase gene expression promoted by the sequence with the ST-LS1 promoter could be observed in E. coli, Yersinia enterocolitica and Agrobacterium tumefaciens. Comparison of the luminescence caused by fusions between luxAB and different promoters on the chromosome and on an endogenous plasmid of Y. enterocolitica demonstrated that the level of the heterologous gene expression caused by the fragment with the ST-LS1 promoter was within the range of gene expression levels caused by endogenous promoters of Y. enterocolitica.     

Bacteriophage Transcription Factor Cro Regulates Virulence Gene Expression in Enterohemorrhagic Escherichia coli

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不同调控元件驱动下菊欧氏杆菌纤维素酶celY基因在大肠杆菌中表达的研究

目的:以菊欧氏杆菌(Erwinia chrysanthemi)基因组DNA为模板,通过PCR方法找到了该菌的β-1,4-内切葡聚糖酶celY基因及其调控元件并克隆至pUC19载体。为提高纤维素酶celY基因在原核细胞中的分泌表达量,比较了由不同启动子和信号肽调控的celY基因在大肠杆菌中的表达水平。方法:分别构建了由脂蛋白启动子、T7启动子、果胶酶信号肽调控的多种表达载体与由纤维素酶celY基因自身的启动子和信号肽调控的表达载体相比较。结果:由脂蛋白启动子、T7启动子、果胶酶信号肽调控的表达载体都不同程度提高了celY基因的分泌表达量。结论:脂蛋白启动子、T7启动子、果胶酶信号肽都不失为构建强分泌表达载体的可选元件。     

Distribution and Abundance of Insertion Sequences among Natural Isolates of Escherichia coli

A reference collection of 71 natural isolates of Escherichia coli (the ECOR collection) has been studied with respect to the distribution and abundance of transposable insertion sequences using DNA hybridization. The data include 1173 occurrences of six unrelated insertion sequences (IS1, IS2, IS3, IS4, IS5 and IS30). The number of insertion elements per strain, and the sizes of DNA restriction fragments containing them, is highly variable and can be used to discriminate even among closely related strains. The occurrence and abundance of pairs of unrelated insertion sequences are apparently statistically independent, but significant correlations result from stratifications in the reference collection. However, there is a highly significant positive association among the insertion sequences considered in the aggregate. Nine branching process models, which differ in assumptions regarding the regulation of transposition and the effect of copy number on fitness, have been evaluated with regard to their fit of the observed distributions. No single model fits all copy number distributions. The best models incorporate no regulation of transposition and a moderate to strong decrease in fitness with increasing copy number for IS1 and IS5, strong regulation of transposition and a negligible to weak decrease in fitness with increasing copy number for IS3, and less than strong regulation of transposition for IS2, IS4 and IS30.     

Expression of the Arginine Regulon of Escherichia coli W: Evidence for a Second Regulatory Gene

Effect of the M (modifier) gene of Escherichia coli W on the expression of wild-type structural genes of four arginine biosynthetic enzymes was studied by examining enzyme activity in cell-free extracts of cultures grown in minimal medium and medium containing arginine. The mutant M gene was originally identified as causing arginine-induced synthesis of acetylornithine delta-transaminase in a strain deficient for the enzyme. The strains used in this study received the mutant M gene by recombination. Noncoordinate repression has been demonstrated for two more enzymes of the arginine regulon of E. coli W and the M(-) gene increases the degree of noncoordinate repression for the regulon. Mutation of the M gene results in altered regulation of acetylornithine delta-transaminase, ornithine transcarbamylase, and acetylornithinase. In addition, a decreased growth rate is observed. It is proposed that the M gene is a regulatory gene. A model is presented to explain the data which involves changes in operator-repressor affinity for the structural genes and possibly for the gene controlling arginyl transfer ribonucleic acid synthetase.     

乙内酰脲水解酶基因在大肠杆菌中的克隆表达

节杆菌BT801的乙内酰脲酶系能够水解5-苄基乙内酰脲生成L-苯丙氨酸,其中乙内酰脲水解酶负责乙内酰脲的水解开环。乙内酰脲水解酶的表达对于乙内酰脲酶的催化机制研究及氨基酸的生物不对称合成都具有重要意义。通过PCR技术扩增得到乙内酰脲水解酶基因(hyuH),置于表达载体pT221的,17启动子下游,将构建的重组质粒引入大肠杆菌BL21(DE3)。SDS-PAGE分析在相对分子量50kD处有一较强的表达带,经薄层扫描分析目的蛋白占全菌蛋白的40％,主要以可溶性形式存在,活性分析表明表达产物具有天然的酶活性。