嗜酸氧化亚铁硫杆菌基因组学研究进展

嗜酸氧化亚铁硫杆菌(Acidithiobacillus ferrooxidan,A.ferrooxidans)广泛存在于酸性矿物废水中,与生物冶金和环境净化紧密相关。不同来源嗜酸氧化亚铁硫杆菌全基因组的测序,为我们利用比较基因组学和功能基因组学的方法去洞察嗜酸氧化亚铁硫杆菌功能基因,提供了坚实的研究基础和丰富的科研信息。简述了嗜酸氧化亚铁硫杆菌基因组学的基本特征;从比较基因组学和功能基因组学发现了嗜酸氧化亚铁硫杆菌菌株基因组水平的差异;通过生物信息学概述了该菌的铁和硫代谢机制,并从细菌的功能基因组学对其在生物冶金与环境治理等应用进行了展望。     

极端嗜盐菌冷冻干燥保藏研究

本文报道了不同浓度的海藻糖和蔗糖作为保护剂对盐生盐杆菌（Ｈａｌｏｂａｃｔｅｒｉｕｍｈａｌｏｂｉｕｍ）Ｒ＿１菌株冷冻干燥存活性的影响。结果表明：６％海藻糖,１２％蔗糖和１５％ＮａＣｌ组成的混合保护剂,是冷冻干燥保藏Ｒ＿１菌株极佳的保护剂。采用该保护刑冷冻干燥２５株极端嗜盐菌,于４℃保藏１４个月,经液体培养基和斜面培养基培养检测,全部存活。     

紫外线和60Co辐照诱变选育高效嗜酸氧化亚铁硫杆菌

依次利用紫外线和60Co-γ射线辐照诱变的方法对嗜酸氧化亚铁硫杆菌进行诱变,选育高效嗜酸氧化亚铁硫杆菌菌株.结果表明,紫外诱变可以有效提高嗜酸氧化亚铁硫杆菌的Fe2+氧化速率,最佳紫外线诱变时间为240 s.诱变后菌株的Fe2+氧化速率从0.273 g/L/h提高到了0.312 g/L/h.继续利用60Co-γ射线进行...     

氧化亚铁硫杆菌在琼脂平板上的生长及其计数

氧化亚铁硫杆菌(Thiobacillus ferrooxidans)是一类专性嗜酸自养细菌,在细菌浸矿中起着重要的作用,它已被广泛应用于有用金属的浸出,特别是铜和铀。由于该菌处理矿石时浸出效果与其     

硫酸铁对嗜酸氧化亚铁硫杆菌铁代谢基因表达的影响

目的:探讨硫酸铁对嗜酸氧化亚铁硫杆菌生长及铁代谢基因表达的影响.方法:用血细胞计数板和重铬酸钾滴定法研究了不同浓度硫酸铁对嗜酸氧化亚铁硫杆菌生长的影响,采用实时荧光定量PCR技术研究了硫酸铁对嗜酸氧化亚铁硫杆菌铁代谢基因表达的影响.结果:硫酸铁对嗜酸氧化亚铁硫杆菌的生长有抑制作用,随着培养基中硫酸铁浓度的增加,抑制作用也越来越明显.实时定量PCR结果表明,用0.15 M硫酸铁刺激细菌后,实验中所涉及的铁代谢相关基因表现出相似的表达趋势.刺激10 min后,基因均发生明显的的表达上调,且表现为最大值.刺激时间延长,上调幅度逐渐减小.不同的操纵子基因表现出不同的上调幅度.结论:硫酸铁对嗜酸氧化亚铁硫杆菌的生长有明显的抑制作用,铁代谢相关基因对硫酸铁的刺激有积极的反应.     

铜蓝精矿的生物浸出

采用两种嗜酸硫杆菌(嗜酸氧化亚铁硫杆菌和喜温硫杆菌)对铜蓝进行生物浸出,实验在有或没有4 g/L硫酸亚铁pH 2．0、150转/分、35℃的三角瓶中进行。实验结果表明:用两种菌混合浸出的铜几乎等于嗜酸氧化亚铁硫杆菌单独浸出的铜;另外,亚铁的加入能提高铜的浸出。     

极端嗜盐菌的保藏研究

研究了在极端嗜盐菌的液氮超低温冻结保藏和真空冷冻干燥保藏中,不同种类的保护剂和保护剂的不同盐浓度对存活性的影响．结果表明：在极端嗜盐菌的保藏中,一定浓度的NaCl是保护剂中不可缺少的组成成分之一,但保护剂的盐浓度没有必要达到或接近生长需要的最适盐浓度（20％—25％NaCl）;在真空冷冻干燥保藏中,用6％的海藻糖代替脱脂牛奶作为保护剂,可以得到较高的细胞存活率。77株嗜盐古细菌保藏12个月后检测,全部存活,其中19株菌保藏24个月,检测结果为全部存活．     

嗜酸氧化亚铁硫杆菌RND家族转运蛋白基因(rnd-1)的克隆及生物信息学分析

RND(resistance-nodulation-division)家族转运蛋白广泛分布于革兰氏阴性细菌中,具有催化胞内活性物质(胆酸盐,脂肪酸等)和外排金属离子(Cu~+,Ag~+)的作用。为探究嗜酸氧化亚铁硫杆菌(Acidithiobaeillus ferrooxidans,A.ferrooxidans)RND transporter的功能特性,以本实验室分离鉴定的A.ferrooxidans GB2为材料,采用PCR扩增技术克隆了rnd-1基因。运用生物信息学在线分析软件对该基因编码的氨基酸序列的理化性质、结构特征、功能结构域、系统发育以及可能参与的代谢通路等进行了预测。数据表明:该基因cDNA序列全长1 320 bp,编码439个氨基酸,分子式为C_(2100)H_(3330)N_(610)O_(604)S_(13),相对分子质量为47.204 kD,为亲水性蛋白;无信号肽,富含延伸链和无规则卷曲结构,三维结构预测表明其符合4KKU结构生物学特点;采用最大似然法构建系统发生树发现RND transporter蛋白在嗜酸氧化亚铁硫杆菌与嗜酸氧化硫硫杆菌中亲缘关系比较近;肽链含有10处磷酸化位点,10处糖基化位点,可能参与细胞质中磷酸化反应,从而对重金属胁迫进行感知和表达。本研究推测该基因在微生物抗重金属过程中有重要的作用。     

降胆固醇益生菌发酵剂的制备

以前期筛选出来的降胆固醇益生菌嗜酸乳杆菌S-59为出发菌株，对冻干发酵剂的制备工艺进行研究和优化。结果表明：菌悬液的离心收集条件为5 000 r/min、20 min；通过单因素试验和响应面优化试验，确定冻干保护剂为脱脂乳8.95%、海藻糖5.24%、甘油4.54%，细胞存活率可达90.1%。冻干发酵剂的保藏条件为在真空条件下-4℃保藏。     

氧化亚铁硫杆菌中磁小体形成相关基因在不同亚铁浓度刺激下的差异表达

氧化亚铁硫杆菌(Acidithiobacillus ferrooxidans)的生物控制矿化作用可以使其在胞内形成黑色电子致密颗粒—磁小体。本研究利用生物信息学方法对氧化亚铁硫杆菌标准菌株ATCC 23270的全基因组进行分析, 并通过Real-time PCR技术研究氧化亚铁硫杆菌中与磁小体形成相关的mpsA、magA、thy和mamB四个基因在不同亚铁浓度刺激下的差异表达, 结果发现它们在转录层面的表达量受亚铁浓度的影响, 当亚铁浓度达到150~200 mmol/L范围内达到最高表达,这对进一步深入研究氧化亚铁硫杆菌中磁小体的形成机理有积极的意义。     

Glycine betaine as a cryoprotectant for prokaryotes

Osmoprotectants are low molecular weight, hydrophilic, nontoxic molecules that assist a cell under osmotic stress to stabilize its concentration of internal solutes. These properties are similar to compounds used as cryoprotectants for the preservation of prokaryotic cells during freezing. This study tested the ability of a common compatible solute, glycine betaine (GB), to act as a cryoprotectant. In a series of freeze-drying studies using a variety of prokaryotes, GB performed as well, or better than, two commonly used cryoprotectants, sucrose/bovine serum albumin (S/BSA) and trehalose/dextran (T/D). GB did especially well maintaining cell viability after long-term storage (simulated equivalent of 20 years) for microorganisms like Neisseria gonorrhoeae and Streptococcus pneumoniae. GB was tested for its ability to preserve members of the genus Acidothiobacillus, a difficult genus to preserve. For two strains of Acidithiobacillus ferrooxidans that were preserved using liquid drying, GB performed as well as S/BSA. Results were more mixed for two strains of Acidithiobacillus thiooxidans; one strain could be preserved with S/BSA but not GB, the other strain gave low recoveries with both cryoprotectants. GB also proved to be a useful cryoprotectant for liquid nitrogen preservation yielding equivalent results to the cryopreservative, glycerol for halophilic archaea, and neutrophilic Fe-oxidizing bacteria. These results indicate that GB is a simple and useful cryoprotectant that works for a wide range of prokaryotic organisms under different cryopreservation regimens.     

响应面法降低肝细胞冷冻保护液中的二甲基亚砜浓度

为有效降低干细胞冷冻保护液中的二甲基亚砜(Me2SO,DMSO)浓度,减少其作为冷冻保护剂对细胞低温保存时产生的毒性,提高细胞存活率,以肝细胞为目标,采取响应面法对冷冻保护剂配方进行了优化。结果表明：冷冻保护剂的最佳配比为DMSO浓度3%、甘油浓度6%、海藻糖浓度0.1%,在此条件下肝细胞复温存活率可达到84.35%,研究结果有效地降低了DMSO的浓度,减少了由此带来的对肝细胞造成的毒性损害,为后续的研究奠定了基础。     

海藻糖对双歧杆菌DM8504菌株冷冻干燥保护效果的研究

本文就海藻糖对ＤＭ８５０４双歧杆菌的冻干保护效果进行了研究,考察了海藻糖浓度、保护剂溶液与菌泥混合时间对ＤＭ８５０４冻干存活率的影响,结果证实该保护剂对ＤＭ８５０４双歧杆菌有良好的冻干保护效果,优于其它保护剂。     

Optimisation of initial cell concentration enhances freeze-drying tolerance of Pseudomonas chlororaphis

The freeze-drying tolerance of Pseudomonas chlororaphis, an antifungal bacterium used as biocontrol agent was investigated. P. chlororaphis is freeze-drying sensitive and the viability drops more than 3 log units in the absence of protective freeze-drying medium. Of the freeze-drying media tested, lactose, sucrose, trehalose, glutamate, sucrose with glutamate, skimmed milk, and skimmed milk with trehalose, skimmed milk gave the lowest survival (0.6+/-0.2%) and sucrose the highest (6.4+/-1.2%). Cellular accumulation of sucrose from the freeze-drying medium and the protective effect of sucrose were dependent on sucrose concentration. The effect of initial cell concentration, from 1 x 10(7) to 5 x 10(10) CFU/ml, on survival after freeze-drying was studied for carbon starved cells with sucrose as freeze-drying medium. The highest freeze-drying survival values, 15-25%, were obtained for initial cell concentrations between 1 x 10(9) and 1 x 10(10) CFU/ml. For cell concentrations outside this window more than 10 times lower survival values were observed. P. chlororaphis was cultivated to induce stress response that could confer protection against freeze-drying inactivation. Carbon starvation and, to a lesser extent, heat treatment enhanced freeze-drying tolerance. By combining optimal cell concentration, optimal sucrose concentration and carbon starvation the survival after freeze-drying was 26+/-6%.     

Comparison of permeating and nonpermeating cryoprotectants for mouse sperm cryopreservation

Mouse sperm has proven to be more difficult to cryopreserve than sperm of other mammalian species. Published reports show that only three cryoprotectant agents (CPAs), alone or combined, have been studied: glycerol and dimethyl sulfoxide (DMSO), as permeating agents, and raffinose, as a nonpermeating agent. To date, the most consistent results for mouse sperm cryopreservation have been achieved by use of raffinose/skim milk as cryoprotectant with rapid cooling at 20 degrees C per minute. In this study, we compared the cryoprotection provided by permeating (glycerol, formamide, propanediol, DMSO, adonitol) or nonpermeating (lactose, raffinose, sucrose, trehalose, d-mannitol) compounds for freezing mouse sperm. Different solutions were made using 3% skim milk solution as the buffer or extender in which all different cryoprotectant agents were dissolved at a concentration of 0.3 M, with a final osmolality of approx. 400 mOsm. Sperm samples from CB6F1 (hybrid) and C57BL/6J (inbred) mice collected directly into each CPA were frozen/thawed under identical conditions. After thawing and CPA elimination (centrifugation) raffinose (59%), trehalose (61%), and sucrose (61%) sustained the best motility (P = < 0.1) of the nonpermeating agents, whereas the best of the permeating agents was DMSO (42%). Membrane integrity was analyzed and showed that the simple exposure (prefreeze) to sugars was less harmful than the exposure to glycols. Coincidentally, sperm frozen in trehalose (41%), raffinose (40.5%), and sucrose (37.5%) were the samples less injured among all different postthawed CPA tested. The in vitro fertilization results demonstrated that hybrid mouse spermatozoa frozen with sugars (lactose 80%, raffinose 80%, trehalose 79% of two-cell embryos production) were more fertile than those frozen with glycols (glycerol 11%).     

Cryopreservation of transgenic rice suspension cells producing recombinant hCTLA4Ig

Transgenic suspension cells of Oryza sativa L. cv. Dongjin utilized as a host for producing recombinant human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) were preserved in liquid nitrogen (−196 °C) after slow prefreezing in a deep freezer (−70 °C). The development of an optimal procedure for long-term storage was investigated by the addition of various concentrations of cryoprotectant mixture and osmoticum in preculture media before cooling. A pre-deep-freezing time of 120 min was the most effective for maintaining cell viability. Compared with mannitol, sorbitol, trehalose, and NaCl under the same osmotic conditions, 0.5 M sucrose was found to be the best osmoticum for preculture media. The cryoprotectant comprising sucrose, glycerol, and dimethylsulfoxide (DMSO) was applied to the precultured cells, and a combination of 1 M sucrose, 1 M glycerol, and 1 M DMSO provided the best result. The viability with this optimized condition was 88% after cryocell-banking for 1 day. The expression of hCTLA4Ig in recovered callus from cryopreservation was also kept stable, and the production level was similar to that observed in noncryopreserved cultures.     

Effect of reduced concentrations of glycerol and various macromolecules on the cryopreservation of mouse and cattle embryos

The effect of different macromolecules [bovine serum albumin (BSA), Pluronic F-68, (ET surfactant), or sodium hyaluronate (SH)] on postthaw survival of mouse morulae and in vivo- and in vitro-derived bovine blastocysts frozen in 10, 5, or 1% glycerol solutions was investigated. Embryos were equilibrated with cryoprotectant solution at 25 degrees C for 10 min, seeded at -5 degrees C, cooled at 0.5 degrees C/min to -35 degrees C, and plunged into liquid nitrogen. Embryos were thawed in a 35 degrees C water bath, glycerol was removed with 0.6 M sucrose at 25 degrees C for 5 min, and postthaw viability was evaluated after 1, 24, and 48 h in culture. The addition of BSA supplementation improved postthaw survival of mouse morulae frozen in 5% glycerol, but not in 10% glycerol. All three macromolecular supplements were effective in increasing survival of mouse morulae in 5% glycerol but only BSA and SH were effective in increasing postthaw survival of in vivo- and in vitro-derived bovine blastocysts. None of the macromolecular supplements improved postthaw survival of embryos frozen in 1% glycerol.     

Stabilization of freeze-dried Lactobacillus paracasei subsp. paracasei JCM 8130T with the addition of disaccharides,polymers, and their mixtures

Although freeze-drying is a widely used dehydration technique for the stabilizing of unstable lactic acid bacteria, Lactobacillus paracasei subsp. paracasei JCM 8130^T (L. paracasei) is destabilized after freeze-drying and subsequent storage. In order to improve the stability of freeze-dried L. paracasei, effects of disaccharides (sucrose and trehalose), polymers (maltodextrin; MD and bovine serum albumin; BSA), and their mixtures on the survival rate of freeze-dried L. paracasei were investigated. The survival rate of non-additive sample decreased slightly after freeze-drying but decreased drastically after subsequent storage at 37 °C for 4 weeks. The reduction was diminished by the addition of disaccharides and polymers. The stabilizing effect of disaccharides was not affected by the co-addition of MD. In contrast, the disaccharide–BSA mixtures had a synergistic stabilizing effect, and the survival rates were largely maintained even after storage. It is suggested that the synergistic effect originates from the conformational stabilization of the dehydrated bacteria.     

Stability of Protein-Encapsulating DRV Liposomes After Freeze-Drying: A Study with BSA and t-PA

Stability of protein-encapsulating DRV (dried-rehydrated vesicle) liposomes is evaluated after freeze-drying vesicles in presence (or not) of trehalose. Two proteins, bovine serum albumin (BSA) and tissue-type plasminogen activator (t-PA), are used, and protein-encapsulating liposomes with different lipid compositions are prepared by DRV technique. Encapsulation efficiencies are calculated, after measuring BSA with a fluorescence technique and t-PA's amidolytic activity toward a chromogenic substrate.

Experimental results show that encapsulation of BSA in vesicles ranges between 35 and 53% of the protein and is only slightly affected by lipid composition. For t-PA, entrapment efficiencies are lower, ranging between 2 and 16%, while lipid composition has substantial effect on entrapment (cholesterol inclusion is very important). After freeze-drying, some lipid compositions remain stable, retaining most of initially entrapped proteins, while others do not, but they may be stabilized by trehalose. In the case of BSA, liposome behavior cannot be explained based on lipid membrane rigidity (more rigid?=?more stable). This may be connected with previously demonstrated interactions of BSA with membranes. Oppositely, t-PA behavior is more predictable, meaning that the lipid composition selected for the specific therapeutic application determines the need for cryoprotectant addition before freeze-drying t-PA containing DRV liposomes, perhaps due to the fact that under conditions applying minimum or no interactions between t-PA and lipid membranes occur.

Thereby, interactions between proteins and membranes determine not only the encapsulation efficiency but also the need for cryopreservation of liposomal protein formulations.     

不同冷冻保护剂对早期卵裂期小鼠胚胎冷冻后成活率和发育的影响

为了评价利用不同冷冻保护剂冷冻早期卵裂期胚胎的效果,用小鼠为实验动物,采用慢速冷冻、快速融解的冷冻技术,比较丙二醇、二甲基亚砜和甘油作冷冻保护剂对小鼠2-细胞、4-细胞、8-细胞胚胎冷冻后胚胎存活率和囊胚形成率的影响。发现以丙二醇和蔗糖为冷冻保护剂冷冻4-细胞、8-细胞胚胎,解冻后胚胎成活率和囊胚形成率显著高于以二甲基亚砜或甘油为冷冻保护剂。结果表明,丙二醇是一种冷冻早期卵裂期小鼠胚胎有效的冷冻保护剂。