湿度和温度对酰化紫膜LB膜M衰减的影响

用闪光动力学光谱仪测量了酰化紫膜ＬＢ膜中Ｍ衰减速率的变化。酰化紫膜ＬＢ膜的衰减无论是悬浮液状态,还是ＬＢ膜中,均比未修饰的要慢。在温度为２０℃时,酰化紫膜ＬＢ随着相对湿度的增加,Ｍ衰减加快。在相对湿度较低时（ＲＨ３４—７５％）,变化较平缓,即Ｍ的衰减加快不明显;在相对湿度较高时（ＲＨ８４—９５％）,Ｍ衰减明显加快。温度的变化则随相对湿度不同而不同。相对湿度较低时,随着温度的升高,Ｍ衰减加快;相对湿度较高时,Ｍ衰减反而减慢。酰化紫膜悬浮液的Ｍ衰减随着温度的升高而明显加快．这说明酰化紫膜ＬＢ膜中ＢＲ水合程度可能是直接影响Ｍ衰减的因素之一。     

The use of a partition locus to increase stability of tryptophan-operon-bearing plasmids in Escherichia coli

The stability of different derivatives of plasmid vectors pBR322 and pACYC184 carrying the tryptophan operon of Escherichia coli was monitored in various media. It was found that in the absence of any special selective pressure, all plasmids were lost from the culture. The stability varied depending both on the orientation of the inserted tryptophan fragment and the growth media used. The pBR322::trp+ plasmids were lost at an average frequency of 0.3 to 0.8% per cell generation, while the pACYC184::trp+ plasmid was lost at a rate higher than 5%. In all cases the whole plasmid was lost at a rate higher than 5%. In all cases the whole plasmid was lost, indicating a high stability of the plasmid::cloned DNA as such. To increase the stability of the cloning vectors, the partition locus of plasmid pSC101 was added to both the pBR322::trp+ and pACYC184::trp+ plasmids. The addition of this gene increased the replicon stability at least 3- to 10-fold, with the pBR322::trp+-par+ plasmids being the most stable. Also in this case, the stability was dependent on the plasmid type and on the growth medium. In no case was there a discoordinate loss of the antibiotic-resistance and tryptophan genes from the vectors.     

Burkholderia xenovorans LB400联苯双加氧酶及突变体对DDTs的降解

DDTs（dichlorodiphenyltrichloroethane,1,1,1-三氯-2,2-双氯苯基乙烷）是一种典型的持久性有机污染物,曾在疟疾防治和农业除虫方面被广泛应用。虽然包括我国在内的很多国家已经禁止使用DDTs,但目前对环境中DDTs的检测发现它仍然广泛存在且具有新的输入源。DDTs的持续存在对近海生态系统和人类健康具有一定危害,因此它所造成的环境污染问题仍然值得关注。由于Rieske型芳香羟化双加氧酶能够起始多种持久性污染物的降解,过去的几十年里一直是芳香化合物降解领域的焦点。[目的] 为探讨联苯双加氧酶对DDTs的降解特性及机制,本研究选取了食异生素伯克霍尔德氏菌LB400（Burkholderia xenovorans）联苯双加氧酶及突变体对p,p''-DDT和o,p''-DDT的降解过程进行研究。[方法] 以BphAE_LB400为亲本,通过两步定点突变将283位的丝氨酸突变为蛋氨酸,获得突变体BphAE_S283M。通过比较亲本酶与突变体对DDTs的催化性能,模拟突变蛋白结构和分子对接等方法,探究其降解特性及机制。[结果] BphAE_LB400和突变体BphAE_S283M都无法降解对位的p,p''-DDT,但突变体BphAE_S283M可以代谢o,p''-DDT并产生2个立体异构体。对接p,p''-DDT的BphAE_LB400和BphAE_S283M的结构分析表明,BphAE_LB400和BphAE_S283M中p,p''-DDT的反应环均不与原晶体结构中的联苯反应环重合。而对接o,p''-DDT的BphAE_S283M的结构分析表明o,p''-DDT的反应环与晶体结构中的联苯反应环距离很近,且2、3位的碳原子与单核铁原子催化中心的距离在0.5 nm以内,此外,BphAE_S283M的催化腔表面积和体积比BphAE_LB400更大,这很可能有助于BphAE_S283M与o,p''-DDT的结合。[结论] 283位氨基酸是影响BphAE_LB400对DDTs的催化代谢能力的关键氨基酸残基,它可以通过调节反应碳原子与催化中心的距离以及催化腔的大小来影响底物特异性。本次研究进一步阐明了283位氨基酸残基的影响机理,为更有效修复DDTs污染提供理论依据和技术支持。     

Differences between bacterial associations with two root types of Vicia faba L

Abstract

Differences between various inherent physiological characteristics of lateral roots and of taproots of faba bean plants (Vicia faba L.) have been described in the literature. The question as to whether distinct bacterial communities inhabit each of those root types calls for further investigation. This question was tackled using aeroponically grown plants, i.e., plants that were grown under conditions as homogeneous as possible. Samples of the apical 5 cm of taproots and of lateral roots were compared. Metabolic fingerprints of root bacterial communities were analyzed using the Biolog® assay. Specificity of colonization of the different root types by specific bacterial taxa was examined by the Real-Time Polymerase Chain Reaction (PCR) method. Root bacterial communities produced distinct metabolic fingerprints for each of the two root types. Herbaspirillum spp. were found to be associated with lateral roots but not with taproots both under non-saline and saline (50 mM NaCl) conditions. No significant differences were found in the abundance of bacteria with respect to either root type or salinity. It is concluded that different root types, even within single root systems, differ not only in their physiological traits but also in their bacterial associations. Such associations might have adaptive advantages.     

Functional and evolutionary analysis of DXL1, a non-essential gene encoding a 1-deoxy-D-xylulose 5-phosphate synthase like protein in Arabidopsis thaliana

The synthesis of 1-deoxy-D-xylulose 5-phosphate (DXP), catalyzed by the enzyme DXP synthase (DXS), represents a key regulatory step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis. In plants DXS is encoded by small multigene families that can be classified into, at least, three specialized subfamilies. Arabidopsis thaliana contains three genes encoding proteins with similarity to DXS, including the well-known DXS1/CLA1 gene, which clusters within subfamily I. The remaining proteins, initially named DXS2 and DXS3, have not yet been characterized. Here we report the expression and functional analysis of A. thaliana DXS2. Unexpectedly, the expression of DXS2 failed to rescue Escherichia coli and A. thaliana mutants defective in DXS activity. Coherently, we found that DXS activity was negligible in vitro, being renamed as DXL1 following recent nomenclature recommendation. DXL1 is targeted to plastids as DXS1, but shows a distinct expression pattern. The phenotypic analysis of a DXL1 defective mutant revealed that the function of the encoded protein is not essential for growth and development. Evolutionary analyses indicated that DXL1 emerged from DXS1 through a recent duplication apparently specific of the Brassicaceae lineage. Divergent selective constraints would have affected a significant fraction of sites after diversification of the paralogues. Furthermore, amino acids subjected to divergent selection and likely critical for functional divergence through the acquisition of a novel, although not yet known, biochemical function, were identified. Our results provide with the first evidences of functional specialization at both the regulatory and biochemical level within the plant DXS family.     

Microsatellite markers uncover cryptic species of Odontotermes (Termitoidae: Termitidae) from Peninsular Malaysia

Termites from the genus Odontotermes are known to contain numerous species complexes that are difficult to tell apart morphologically or with mitochondrial DNA sequences. We developed markers for one such cryptic species complex, that is, Odontotermes srinakarinensis sp. nov. from Maxwell Hill Forest Reserve (Perak, Malaysia), and characterised them using a sample of 41 termite workers from three voucher samples from the same area. We then genotyped 150 termite individuals from 23 voucher samples/colonies of this species complex from several sites in Peninsular Malaysia. We analysed their population by constructing dendograms from the proportion of shared-alleles between individuals and genetic distances between colonies; additionally, we examined the Bayesian clustering pattern of their genotype data. All methods of analysis indicated that there were two distinct clusters within our data set. After the morphologies of specimens from each cluster were reexamined, we were able to separate the two species morphologically and found that a single diagnostic character found on the mandibles of its soldiers could be used to separate the two species quite accurately. The additional species in the clade was identified as Odontotermes denticulatus after it was matched to type specimens at the NHM London and Cambridge Museum of Zoology.     

Characterisation of a thermo-alkali-stable lipase from oil-contaminated soil using a metagenomic approach

Lipases are widely used for a variety of biotechnological applications. Screening these industrial enzymes directly from environmental microorganisms is a more efficient and practical approach than conventional cultivation-dependent methods. Combined with activity-based functional screening, six clones with lipase activity were detected and a gene (termed lipZ01) isolated from a target clone with the highest lipase activity was cloned from an oil-contaminated soil-derived metagenomic library and then sequenced. Gene lipZ01 was expressed in Pichia pastoris GS115 and the molecular weight of the recombinant lipase LipZ01 was estimated by electrophoresis analysis to be approximately 50 kDa. The maximum activity of the purified lipase was 42 U/mL, and the optimum reaction temperature and pH value were 45 °C and 8.0, respectively. The enzyme was highly stable in the temperature range 35–60 °C and under alkaline conditions (pH 7–10). The presence of Ca²⁺ and Mn²⁺ ions could significantly enhance the activity of the lipase. The purified lipase preferentially hydrolysed triacylglycerols with acyl chain lengths ≥8 carbon atoms, and the conversion degree of biodiesel production was nearly 92% in a transesterification reaction using olive oil and methanol. Some attractive properties suggested that the recombinant lipase may be valuable in industrial applications.