Improvement of pCVD442, a suicide plasmid for gene allele exchange in bacteria

Allelic exchange experiments allow investigation of the functions of many unknown genes identified during the sequencing of entire genomes. Isogenic strains differing by only specific mutations can be constructed. Among other tools, suicide plasmids are widely used for this task. They present many advantages because they leave no scars on the chromosome, and therefore allow combining several mutations in the same genetic background. While using the previously described pCVD442 suicide plasmid [Infect. Immun. 59 (1991) 4310], we found untargeted recombination events due to the presence of an IS1 element on this plasmid. The plasmid was therefore improved by removal of the IS1 element. We also replaced the bla gene of pCVD442, conferring ampicillin resistance, by the cat gene conferring chloramphenicol resistance, leading to the new suicide plasmid pDS132. The plasmid was entirely sequenced. We demonstrate that this new vector can be easily used to introduce various types of mutations into different genetics backgrounds: removal of IS elements, introduction of point mutations or deletions. It can be introduced into bacterial strains by either transformation or conjugation.     

Isoniazid and thioacetazone may exhibit antitubercular activity by binding directly with the active site of mycolic acid cyclopropane synthase: Hypothesis based on computational analysis

Isoniazid and thioacetazone are the two important antitubercular drugs. In case of thioacetazone it is established that it inhibits mycolic acid cyclopropane synthase but the exact binding site accounting for such inhibition is presently unknown. In case of isoniazid its action on the said enzyme is unexplored. In this work we have analyzed the binding of isoniazid and thioacetazone with mycolic acid cyclopropane synthase (CmaA1 and CmaA2) using tools of computational biology. We have observed that thioacetazone fits well at the active site of CmaA1 and CmaA2 while isoniazid binds at the active site of CmaA1 only. We have recommended experimental validation of such results. If such results are proved to be fact it will explore the exact binding site of thioacetazone and discover a new mechanism of anti-tubercular action of isoniazid.     

快速方法检测结核分枝杆菌耐药基因突变

快速准确地鉴定结核分枝杆菌与结核分枝杆菌对利福平和异烟肼耐药基因突变的快速检测,对结核病人的诊断与治疗具有重要指导意义。本次根据结核分枝杆菌标准株H37RV序列,利用覆盖rpoB、katG、inhA基因突变区的系列寡核苷酸探针,并检测临床样品中结核分枝杆菌的基因突变情况,以此来判断耐药结果,并对其进行方法学评价。     

Bioassays for Monitoring Insecticide Resistance

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products are labeled for an individual pest within a particular crop system, chemical control options are limited. Therefore, the same product(s) are used repeatedly and continual selection pressure is placed on the target pest. There are both financial and environmental costs associated with the development of resistant populations. The cost of pesticide resistance has been estimated at approximately $ 1.5 billion annually in the United States. This paper will describe protocols, currently used to monitor arthropod (specifically insects) populations for the development of resistance. The adult vial test is used to measure the toxicity to contact insecticides and a modification of this test is used for plant-systemic insecticides. In these bioassays, insects are exposed to technical grade insecticide and responses (mortality) recorded at a specific post-exposure interval. The mortality data are subjected to Log Dose probit analysis to generate estimates of a lethal concentration that provides mortality to 50% (LC₅₀) of the target populations and a series of confidence limits (CL''s) as estimates of data variability. When these data are collected for a range of insecticide-susceptible populations, the LC₅₀ can be used as baseline data for future monitoring purposes. After populations have been exposed to products, the results can be compared to a previously determined LC₅₀ using the same methodology.     

Drug resistance mechanisms and novel drug targets for tuberculosis therapy

Drug-resistant tuberculosis (TB) poses a significant challenge to the successful treatment and control of TB worldwide. Resistance to anti-TB drugs has existed since the beginning of the chemotherapy era. New insights into the resistant mechanisms of anti-TB drugs have been provided. Better understanding of drug resistance mechanisms helps in the development of new tools for the rapid diagnosis of drug-resistant TB. There is also a pressing need in the development of new drugs with novel targets to improve the current treatment of TB and to prevent the emergence of drug resistance in Mycobacterium tuberculosis. This review summarizes the anti-TB drug resistance mechanisms, furnishes some possible novel drug targets in the development of new agents for TB therapy and discusses the usefulness using known targets to develop new anti-TB drugs. Whole genome sequencing is currently an advanced technology to uncover drug resistance mechanisms in M. tuberculosis. However, further research is required to unravel the significance of some newly discovered gene mutations in their contribution to drug resistance.     

Insights into β-Lactamases from Burkholderia Species,Two Phylogenetically Related yet Distinct Resistance Determinants

Burkholderia cepacia complex and Burkholderia pseudomallei are opportunistic human pathogens. Resistance to β-lactams among Burkholderia spp. is attributable to expression of β-lactamases (e.g. PenA in B. cepacia complex and PenI in B. pseudomallei). Phylogenetic comparisons reveal that PenA and PenI are highly related. However, the analyses presented here reveal that PenA is an inhibitor-resistant carbapenemase, most similar to KPC-2 (the most clinically significant serine carbapenemase), whereas PenI is an extended spectrum β-lactamase. PenA hydrolyzes β-lactams with k_cat values ranging from 0.38 ± 0.04 to 460 ± 46 s⁻¹ and possesses high k_cat/k_inact values of 2000, 1500, and 75 for β-lactamase inhibitors. PenI demonstrates the highest k_cat value for cefotaxime of 9.0 ± 0.9 s⁻¹. Crystal structure determination of PenA and PenI reveals important differences that aid in understanding their contrasting phenotypes. Changes in the positioning of conserved catalytic residues (e.g. Lys-73, Ser-130, and Tyr-105) as well as altered anchoring and decreased occupancy of the deacylation water explain the lower k_cat values of PenI. The crystal structure of PenA with imipenem docked into the active site suggests why this carbapenem is hydrolyzed and the important role of Arg-220, which was functionally confirmed by mutagenesis and biochemical characterization. Conversely, the conformation of Tyr-105 hindered docking of imipenem into the active site of PenI. The structural and biochemical analyses of PenA and PenI provide key insights into the hydrolytic mechanisms of β-lactamases, which can lead to the rational design of novel agents against these pathogens.     

糖尿病合并肺结核患者诱导耐药性危险因素的回归分析

目的:研究2型糖尿病(Type 2 Diabetes,T2DM)合并肺结核(Tuberculosis,TB)患者诱导耐药性危险因素的回归分析。方法:从2012年3月到2013年3月,于我院共计有124例患者被确诊为肺结核,将其作为研究对象。根据患者是否合并有2型糖尿病,将其分成观察组(49例)及对照组(75例)。对全部患者进行耐药性实验,分别经单因素分析及Logistic回归性分析寻找诱导耐药性的危险因素。结果:观察组在治疗过程中断、有吸烟习惯、依从性差、病程≥1年、HbAlc值≥6.5%等方面所占比例显著高于对照组,差异均有统计学意义(均P0.05)。由多因素分析可知,治疗过程中断、有吸烟习惯、依从性差、病程≥1年、HbAlc值≥6.5%等均为糖尿病合并肺结核患者的危险因素。结论:T2DM合并TB患者诱导耐药性的危险因素较多,临床应重点关注,并采取相应措施,从而为临床治疗提供更为有利的条件。     

Tolerance to dry bubble disease (Lecanicillium fungicola) in Iranian wild germplasm of button mushroom (Agaricus bisporus)

Agaricus bisporus is the most widely cultivated mushroom. The mushroom crop is subjected to several fungal diseases. Dry bubble disease caused by Lecanicillium fungicola is among notorious diseases of A. bisporus. This study aimed to assess phenotypic resistance to dry bubble disease among A. bisporus wild strains, collected from Iran regions. The reliability of resistance evaluations regarding disease incidence and intensity was well documented. The extraordinary tolerance of some wild strains to even high degrees of inoculum concentrations (10⁷ and 10⁸ spore/m² mushroom growth bed) of the pathogen in compare to commercial cultivars approved potentials of the wild germplasm in breeding programs for resistance. Also, the potential of some Microsatellite loci for the molecular-based rapid screening of tolerance was established by attributing SSR loci of phenotypically tolerant strains to QTLs for dry-bubble resistance-related traits.     

Actinobacillus pleuropneumoniae serotype 7 siderophore receptor FhuA is not required for virulence

A ferrichrome receptor, FhuA, was identified in Actinobacillus pleuropneumoniae serotype 7. An isogenic mutant with a deletion in the ferrichrome uptake receptor gene (fhuA) was constructed and examined in an aerosol infection model. The disease caused by the mutant was indistinguishable from disease induced by A. pleuropneumoniae serotype 7 wild-type; an isogenic mutant lacking expression of the exbB gene that is required for the uptake of transferrin-bound iron retained the ability to utilize ferrichrome, thereby indicating that an energy-coupling mechanism involved in ferrichrome transport remains to be identified.     

黄淮海北部地区大豆育成品种对黄淮海主要SMV流行株系的抗性评价

采用人工接种方法研究了166份黄淮海北部地区近年来生产上种植品种及近期育成品种(系)对SMV的抗性,利用黄淮海地区SMV流行株系SC3、SC6、SC7、SC11以及混合4个株系进行了抗性鉴定,从客观上评价了上述品种(系)的抗性。结果显示:对4个株系均表现抗病(中抗、高抗和免疫)的共82份,占49.4%;其中对3个株系表现高抗或免疫的32份,占19.3%;对4个株系表现高抗或免疫的23份,占13.9%。对混合株系表现抗病的108份,占65.1%。其中表现免疫的45份,占27.1%,表现高抗的29份,占17.5%。对4个株系和混合株系均表现抗病的62份,占37.3%;表现免疫和高抗的14份,占8.4%。近年育成品种对SC3、SC7株系较早期育成品种的抗性显著增强;来自河北、北京、山西的品种抗性较好,病情指数整体较低。鉴定筛选出对接种的4个株系及混合株系均表现免疫的品种冀豆9号和石豆6号,可作为抗病育种的重要抗源。本研究还发现部分品种对接种的4个株系和混合株系表现出抗性差异,表明SMV株系间存在着明显的互作。     

新型抗结核活性化合物H37Ra的耐药菌筛选及其菌落表型

ATB-152E和ATB-152J为本实验室前期研究获得的具有良好抗结核活性的两种结构类似的小分子化合物,本文就其作用靶标及耐药机制进行探索。采用含药平板涂板筛选以及平板划线培养法逐步提高化合物浓度,分别筛选出结核分枝杆菌ATB-152E和ATB-152J耐药菌株。选取有代表性的耐药菌株,用微孔法测定结核分枝杆菌的最小抑菌浓度,分别对它们的菌落形态、生物膜形成等表型进行观察并与野生株进行比较。通过不断提高化合物筛选浓度最终筛选到ATB-152E耐药菌株17株、ATB-152J耐药菌株15株,这两种化合物的耐药频率均为10^－7。生长表型结果显示,与野生株结核分枝杆菌相比,ATB-152E耐药菌菌落褶皱变多,ATB-152J耐药菌菌落形态更为扁平,褶皱变少。耐药菌的生物膜形成所需时间与野生株也存在差异,提示活性化合物耐药菌的突变可能导致细菌脂质代谢异常。ATB-152E和ATB-152J耐药菌的获得,为后续深入探索这两种具有良好抗结核活性化合物的作用机制奠定了基础。     

Physiological aspects of aluminium tolerance associated with the long arm of chromosome 2D of the wheat (Triticum aestivum L.) genome

Aluminum (Al) uptake in roots of wheat nearisogenic lines having differing tolerances to aluminium toxicity was studied using roots and root segments immersed in a nutrient solution at a controlled pH and temperature. At low Al concentrations a mechanism preventing root tips from accumulating too much Al was observed in an Al-tolerant isoline and a BH1146 euploid. This mechanism was more efficient when divalent cations of calcium or magnesium were present in the nutrient medium. Al accumulation steadily increased in root tips of the Al-sensitive wheat isoline during all 24 h of incubation, and the presence of divalent cations in the medium even increased Al concentration in root tissue. However, at higher Al concentrations in the medium the mechanism preventing the root tips of Al-tolerant genotypes from accumulating too much Al was not observed, and in effect Al concentration in root tips of both Al-tolerant and Al-sensitive isolines increased. It is concluded that genetical factors are located on the long arm of chromosome 2D from the BH1146 euploid that control the mechanism preventing root apical meristems from accumulating too much Al at low Al concentrations in the medium. However, there must be other genetical factors also located on this chromosome segment that control Al detoxication in root tips of Al-tolerant lines at higher external Al concentrations.     

中国春小麦株高、育性近等基因系的建立及应用

以矮败小麦和中国春小麦为材料,经过杂交和连续回交,得到了中国春小麦遗传背景的分别表现矮秆不育、矮秆可育、高秆不育、高秆可育的近等基因系。根据近等基因系各成员系的株高表现,计算出矮秆基因Rht10的降秆强度是69.8%。借助于赤霉酸处理,在幼芽期就可分出矮败中国春小麦后代的不育株与可育株。Abstract：　Use Dwarfing Male-sterile Wheat and cv. Chinese Spring as parents, after cross and continuously back cross, the isogenic lines with Chinese Spring background were developed. These lines include dwarfing male-sterile line, dwarfing fertile line, tall male-sterile line and tall fertile line. The dwarfing intensity of gene Rht10 was calculated to be 69.8% according the differences between the isogenic lines. Treated with GA3solution, the male-sterile and fertile plants in Chinese Spring Dwarfing Male-sterile Wheat can be identified clearly when they are seedlings.