Genetically Modified Vibrio harveyi Strains as Potential Bioindicators of Mutagenic Pollution of Marine Environments

For biodetection of mutagenic pollution of marine environments, an organism naturally occurring in these habitats should be used. We found that marine bacterium Vibrio harveyi may be an appropriate bioindicator of mutagenic pollution. For positive selection of mutants, we developed a simple method for isolation of V. harveyi mutants resistant to neomycin. We constructed genetically modified V. harveyi strains that produce significantly more neomycin-resistant mutants upon treatment with low concentrations of mutagens than the wild-type counterpart. The sensitivity of the mutagenicity test with the V. harveyi strains is at least comparable to (if not higher than) that of the commonly used Ames test, which uses Salmonella enterica serovar Typhimurium strains. Therefore, we consider that the V. harveyi strains described in this report could be used as potential bioindicators of mutagenic pollution of marine environments.     

多功能农药降解基因工程菌株m-CDS-1环境释放安全评价

[目的]为了评价多功能农药降解基因工程菌株m-CDS-1的环境释放中间试验水平的安全性.[方法]通过农药检测、平板计数、Most probable number-PCR(MPN-PCR)和Denaturing Gradient GelElectrophoresis(DGGE)等方法在江苏大丰进行了工程菌株m-CDS-1田间降解农药效果、定殖动态和对土壤微生物群落结构影响的研究.[结果]投加1.01×107CFU/g干土的工程菌株m-CDS-1在30 d时均能完全降解10.71 mg/kg的甲基对硫磷和1.29 mg/kg的呋喃丹.平板计数表明工程菌株m-CDS-1在土壤中快速下降;MPN-PCR检测结果显示,在4 d,15 d和30 d时,0～10 cm混合土壤中该工程菌株的数目分别为2.15±0.98×106CFU/g干土,3.70±4.66×106 CFU/g干土和检测不出.工程菌株m-CDS-1的投加不会对土壤可培养三大微生物菌群数量产生显著影响;基于细菌16S rDNA V3区的DGGE分析结果表明,施加农药对细菌菌落结构有显著影响,4 d,11 d,30 d时农药施用区与空白对照区的图谱相似性分别为17.16%,49.81%和75.01%,但到60 d时的相似性为98.62%.工程菌株m-CDS-1释放在前期对细菌群落结构有一定影响,4 d,11 d和30 d工程菌株释放区相对于空白的相似性分别为49.57%,38.30%和83.30%.在60 d时,空白、施药和施菌小区的图谱相似性都在90%以上.[结论]工程菌株m-CDS-1不仅可同时高效降解甲基对硫磷和呋喃丹,仍保持了实验室内的原有特性,而且不会成为优势菌群长期在土壤环境中存在,也不会对土壤微生物群落结构造成长期影响.     

Genetically Modified Bacterial Strains and Novel Bacterial Artificial Chromosome Shuttle Vectors for Constructing Environmental Libraries and Detecting Heterologous Natural Products in Multiple Expression Hosts

The enormous diversity of uncultured microorganisms in soil and other environments provides a potentially rich source of novel natural products, which is critically important for drug discovery efforts. Our investigators reported previously on the creation and screening of an Escherichia coli library containing soil DNA cloned and expressed in a bacterial artificial chromosome (BAC) vector. In that initial study, our group identified novel enzyme activities and a family of antibacterial small molecules encoded by soil DNA cloned and expressed in E. coli. To continue our pilot study of the utility and feasibility of this approach to natural product drug discovery, we have expanded our technology to include Streptomyces lividans and Pseudomonas putida as additional hosts with different expression capabilities, and herein we describe the tools we developed for transferring environmental libraries into all three expression hosts and screening for novel activities. These tools include derivatives of S. lividans that contain complete and unmarked deletions of the act and red endogenous pigment gene clusters, a derivative of P. putida that can accept environmental DNA vectors and integrate the heterologous DNA into the chromosome, and new BAC shuttle vectors for transferring large fragments of environmental DNA from E. coli to both S. lividans and P. putida by high-throughput conjugation. Finally, we used these tools to confirm that the three hosts have different expression capabilities for some known gene clusters.     

优良面包酵母菌株的杂交育种

本文对实验室保存的面包酵母进行筛选,以不加糖面团发酵力最高的菌株BY-14和高糖面团发酵力最高的菌株BY-6作为两杂交亲本.二倍体菌株经过单倍体制备、分离和筛选后,利用群体杂交方法,获得了一株兼备两亲本优良性能的面包酵母菌株,其不加糖面团发酵力达到菌株BY-14水平,且高糖面团发酵力比菌株BY-6提高了25%.     

Risk Assessment of Genetically Modified Lactic Acid Bacteria Using the Concept of Substantial Equivalence

The use of food-grade microorganisms such as lactic acid bacteria (LAB) is one of the most promising methods for delivering health promoting compounds. Since it is not always possible to obtain strains that have the ability to produce specific compounds naturally or that produce them in sufficient quantities to obtain physiological responses, genetic modifications can be performed to improve their output. The objective of this study was to evaluate if previously studied genetically modified LAB (GM-LAB), with proven in vivo beneficial effects, are just as safe as the progenitor strain from which they were derived. Mice received an elevated concentration of different GM-LAB or the native parental strain from which they were derived during a prolonged period of time, and different health parameters were evaluated. Similar growth rates, hematological values, and other physiological parameters were obtained in the animals that received the GM-LAB compared to those that were fed with the native strain. These results demonstrate that the GM-LAB used in this study are just as safe as the native strains from which they were derived and thus merit further studies to include them into the food chain.     

Survival of Ice Nucleation-Active and Genetically Engineered Non-Ice-Nucleating Pseudomonas syringae Strains after Freezing

The survival after freezing of ice nucleation-active (INA) and genetically engineered non-INA strains of Pseudomonas syringae was compared. Each strain was applied to oat seedlings and allowed to colonize for 3 days, and the plants were subjected to various freezing temperatures. Plant leaves were harvested before and after freezing on two consecutive days, and bacterial populations were determined. Populations of the INA wild-type strain increased 15-fold in the 18 h after the oat plants incurred frost damage at −5 and −12°C. Plants colonized by the non-INA strain were undamaged at −5°C and exhibited no changes in population size after two freeze trials. As freezing temperatures were lowered (−7, −9, and −12°C), oat plants colonized by the non-INA strain suffered increased frost damage concomitant with bacterial population increases following 18 h. At −12°C, both strains behaved identically. The data show a relationship between frost damage to plants and increased bacterial population size during the following 18 h, indicating a potential competitive advantage of INA strains of P. syringae over non-INA strains in mild freezing environments.     

主要转基因作物研究现状及其产业化进展

经过12年的产业化发展,转基因作物研究取得了较大进展。相关外源基因的转入使抗虫、抗病、抗除草剂、抗逆、品质改良和功能性等作物相继问世。转基因大豆、玉米、棉花和油菜已经大面积种植,产生了极大的经济效益。2007年全球转基因作物的种植面积达到1．143亿hm^2。综述了主要转基因作物研究现状及其产业化进展,并对我国转基因作物产业化应解决的问题进行了论述。     

Comparative Analysis of Geometry and Electrical Properties of Simulated Normal and Genetically Modified Motoneurons

In computer models of spinal motoneurons with reconstructed dendritic arborizations, we performed a comparative study of the features of the dendritic morphology and those of the output discharge patterns generated in response to stepwise or triangular ramp depolarizing currents applied to the soma; the modeled motoneurons were taken from wild-type (WT) and mutant Cu/Zn superoxide dismutase 1 (SOD1) animals. Six dendrites of the SOD1 motoneuron formed a more complex arborization than a 10-dendrite branching of the WT cell. Although both arborizations had similar maximum path distances of about 600 μm, a maximum dendritic complexity of the SOD1 cell was found at path distances of about 300 μm from the soma (about 100 μm farther than that of the WT cell). A greater number of dendritic paths terminating at longer path distances from the soma formed also different morphometrical asymmetry patterns of SOD1 arborizations, as compared with those of WT ones. The model neurons were capable of reproducing several types of experimentally recorded discharge patterns described in the literature. Main differences between the two cells were observed in the hysteretic frequency-current relations characterizing the discharges caused by the ramp current. Compared to the WT motoneuron, the SOD1 cell (i) had a wider dynamic range of the current intensities at which the discharge was initiated and terminated, (ii) this range was shifted to greater current intensities, and (iii) the maximum discharge frequency was lower. The nature of the found structure-related differences still has to be explored and interpreted considering that SOD1 mutants mimic some cases of amyotrophic lateral sclerosis.     

Genetic Basis of Variations in Nitrogen Source Utilization in Four Wine Commercial Yeast Strains

The capacity of wine yeast to utilize the nitrogen available in grape must directly correlates with the fermentation and growth rates of all wine yeast fermentation stages and is, thus, of critical importance for wine production. Here we precisely quantified the ability of low complexity nitrogen compounds to support fast, efficient and rapidly initiated growth of four commercially important wine strains. Nitrogen substrate abundance in grape must failed to correlate with the rate or the efficiency of nitrogen source utilization, but well predicted lag phase length. Thus, human domestication of yeast for grape must growth has had, at the most, a marginal impact on wine yeast growth rates and efficiencies, but may have left a surprising imprint on the time required to adjust metabolism from non growth to growth. Wine yeast nitrogen source utilization deviated from that of the lab strain experimentation, but also varied between wine strains. Each wine yeast lineage harbored nitrogen source utilization defects that were private to that strain. By a massive hemizygote analysis, we traced the genetic basis of the most glaring of these defects, near inability of the PDM wine strain to utilize methionine, as consequence of mutations in its ARO8, ADE5,7 and VBA3 alleles. We also identified candidate causative mutations in these genes. The methionine defect of PDM is potentially very interesting as the strain can, in some circumstances, overproduce foul tasting H₂S, a trait which likely stems from insufficient methionine catabolization. The poor adaptation of wine yeast to the grape must nitrogen environment, and the presence of defects in each lineage, open up wine strain optimization through biotechnological endeavors.     

Indigenous Microbiota and Habitat Influence Escherichia coli Survival More than Sunlight in Simulated Aquatic Environments

The reported fate of Escherichia coli in the environment ranges from extended persistence to rapid decline. Incomplete understanding of factors that influence survival hinders risk assessment and modeling of the fate of fecal indicator bacteria (FIB) and pathogens. FIB persistence in subtropical aquatic environments was explored in outdoor mesocosms inoculated with five E. coli strains. The manipulated environmental factors were (i) presence or absence of indigenous microbiota (attained by natural, disinfected, and cycloheximide treatments), (ii) freshwater versus seawater, and (iii) water column versus sediment matrices. When indigenous microbes were removed (disinfected), E. coli concentrations decreased little despite exposure to sunlight. Conversely, under conditions that included the indigenous microbiota (natural), significantly greater declines in E. coli occurred regardless of the habitat. The presence of indigenous microbiota and matrix significantly influenced E. coli decline, but their relative importance differed in freshwater versus seawater. Cycloheximide, which inhibits protein synthesis in eukaryotes, significantly diminished the magnitude of E. coli decline in water but not in sediments. The inactivation of protozoa and bacterial competitors (disinfected) caused a greater decline in E. coli than cycloheximide alone in water and sediments. These results indicate that the autochthonous microbiota are an important contributor to the decline of E. coli in fresh and seawater subtropical systems, but their relative contribution is habitat dependent. This work advances our understanding of how interactions with autochthonous microbiota influence the fate of E. coli in aquatic environments and provides the framework for studies of the ecology of enteric pathogens and other allochthonous bacteria in similar environments.     

转基因作物非预期效应与自然变异的比较和鉴别

筛查转基因作物非预期效应遇到的最大难题是难以鉴别其非预期效应和自然变异.从转基因作物非预期效应与自然变异的发生机制、食用安全风险和遗传特性的比较分析阐述两者在发生机制及遗传特性方面的差异和特征,为设计减少自然变异干扰的非预期效应筛查模式和方案提供重要启示.     

Gene Expression Analysis of Cold and Freeze Stress in Baker's Yeast

We used mRNA differential display to assess yeast gene expression under cold or freeze shock stress conditions. We found both up- and down-regulation of genes, although repression was more common. We identified and sequenced several cold-induced genes exhibiting the largest differences. We confirmed, by Northern blotting, the specificity of the response for TPI1, which encodes triose-phosphate isomerase; ERG10, the gene for acetoacetyl coenzyme A thiolase; and IMH1, which encodes a protein implicated in protein transport. These genes also were induced under other stress conditions, suggesting that this cold response is mediated by a general stress mechanism. We determined the physiological significance of the cold-induced expression change of these genes in two baker's yeast strains with different sensitivities to freeze stress. The mRNA level of TPI1 and ERG10 genes was higher in freeze-stressed than in control samples of the tolerant strain. In contrast, both genes were repressed in frozen cells of the sensitive strain. Next, we examined the effects of ERG10 overexpression on cold and freeze-thaw tolerance. Growth of wild-type cells at 10°C was not affected by high ERG10 expression. However, YEpERG10 transformant cells exhibited increased freezing tolerance. Consistent with this, cells of an erg10 mutant strain showed a clear phenotype of cold and freeze sensitivity. These results give support to the idea that a cause-and-effect relationship between differentially expressed genes and cryoresistance exists in Saccharomyces cerevisiae and open up the possibility of design strategies to improve the freeze tolerance of baker's yeast.     

Environmental Justice and Risk Assessment: The Uneasy Relationship

Allegations that disproportionate environmental risks fall on low-income and minority communities promote calls for “environmental justice.” A related claim suggests that higher rates of some diseases stem from unequal risks. The empirical evidence supporting these claims remains weak, but uncertainty and controversy are unlikely to abate in the near future. The environmental justice movement has successfully mobilized its constituents, and captured the attention of policymakers, with a politically potent rhetoric of “risk and racism.” Ironically, the movement remains largely uninterested in, or even hostile to, formal risk assessment even while ostensibly calling for more of it.     

Effects of GPD1 Overexpression in Saccharomyces cerevisiae Commercial Wine Yeast Strains Lacking ALD6 Genes

The utilization of Saccharomyces cerevisiae strains overproducing glycerol and with a reduced ethanol yield is a potentially valuable strategy for producing wine with decreased ethanol content. However, glycerol overproduction is accompanied by acetate accumulation. In this study, we evaluated the effects of the overexpression of GPD1, coding for glycerol-3-phosphate dehydrogenase, in three commercial wine yeast strains in which the two copies of ALD6 encoding the NADP⁺-dependent Mg²⁺-activated cytosolic acetaldehyde dehydrogenase have been deleted. Under wine fermentation conditions, the engineered industrial strains exhibit fermentation performance and growth properties similar to those of the wild type. Acetate was produced at concentrations similar to that of the wild-type strains, whereas sugar was efficiently diverted to glycerol. The ethanol yield of the GPD1 ald6 industrial strains was 15 to 20% lower than that in the controls. However, these strains accumulated acetoin at considerable levels due to inefficient reduction to 2,3-butanediol. Due to the low taste and odor thresholds of acetoin and its negative sensorial impact on wine, novel engineering strategies will be required for a proper adjustment of the metabolites at the acetaldehyde branch point.     

The Use of Epidemiology in Environmental Risk Assessment

Epidemiology provides estimates of the concentration–response relation for environmental and occupational toxicants in the species of interest, in or close to the dose range of interest. As such, when available, they provide the primary source for risk assessments. Further information can be acquired by using modern biostatistical techniques to assess the shape of the dose response relation, examine effect modification, and assure control for confounding. These approaches are particularly effective if they are done in the context of a meta-analysis or hierarchical model. This is illustrated with examples from the air pollution literature.