Properties of the Escherichia coli rhodanese-like protein SseA: contribution of the active-site residue Ser240 to sulfur donor recognition.

The product of Escherichia coli sseA gene (SseA) was the subject of the present investigation aimed to provide a tool for functional classification of the bacterial proteins of the rhodanese family. E. coli SseA contains the motif CGSGVTA around the catalytic cysteine (Cys238). In eukaryotic sulfurtransferases this motif discriminates for 3-mercaptopyruvate:cyanide sulfurtransferase over thiosulfate:cyanide sulfurtransferases (rhodanese). The biochemical characterization of E. coli SseA allowed the identification of the first prokaryotic protein with a preference for 3-mercaptopyruvate as donor substrate. Replacement of Ser240 with Ala showed that the presence of a hydrophobic residue did not affect the binding of 3-mercaptopyruvate, but strongly prevented thiosulfate binding. On the contrary, substitution of Ser240 with an ionizable residue (Lys) increased the affinity for thiosulfate.     

Membrane topology and assembly of the outer membrane protein OmpA of Escherichia coli K12

The 325-residue outer membrane protein OmpA of Escherichia coli has been proposed to consist of a membrane-embedded moiety (residues 1 to about 170) and a C-terminal periplasmic region. The former is thought to comprise eight transmembrane segments in the form of antiparallel -strands, forming an amphiphilic connected by exposed turns. Several questions concerning this model were addressed. Thus no experimental evidence had been presented for the turns at the inner leaflet of the membrane and it was not known whether or not the periplasmic part of the polypeptide plays a role in the process of membrane incorporation. Oligonucleotides encoding trypsin cleavage sites were inserted at the predicted turn sites of the ompA gene and it was shown that the encoded proteins indeed become accessible to trypsin at the modified sites. Together with previous results, these data also show that the turns on both sides of the membrane do not possess specifically topogenic information. In two cases one of the two expected tryptic fragments was lost and could be detected at low concentration in only one case. Therefore, bilateral proteolytic digestion of outer membranes can cause loss of -strands and does not necessarily produce a reliable picture of protein topology. When ompA genes were constructed coding for proteins ending at residue 228 or 274, the membrane assembly of these proteins was shown to be partially defective with about 20% of the proteins not being assembled. No such defect was observed when, following the introduction of a premature stop codon, a truncated protein was produced ending with residue 171. It is concluded that (1) the proposed -barrel structure is essentially correct and (2) the periplasmic part of OmpA does not play an active role in, but can, when present in mutant form, interfere with membrane assembly.     

和厚朴酚对大肠埃希氏菌生物被膜形成的抑制机制

【目的】研究不同浓度的和厚朴酚(honokiol)抑制大肠埃希菌(Escherichia coli)的供试菌株10389生物被膜(biofilm,BF)形成的作用机制。【方法】用氯化三苯基四氮唑比色法(TTC)和四唑盐减低法(XTT)测定honokiol抑制E.coli10389生物被膜形成的药物最低抑菌浓度(MIC)和最低杀菌浓度(MBC)及其抑制作用与时间的关系;通过qRT-PCR法检测不同浓度的honokiol对E. coli 10389生物被膜形成基因和群体感应系统相关基因表达量的影响;通过生物发光法和qRT-PCR法检测亚-MIC honokiol对E. coli 10389呋喃糖基硼酸二酯(AI-2)及其调控的与生物被膜形成相关的下游基因表达量的影响。【结果】Honokiol能抑制E.coli10389生物被膜的形成,但不同浓度的honokiol抑制E. coli 10389 BF形成的作用机制不同。其中,与对照组相比,MIC的honokiol能使E. coli 10389 BF形成相关基因编码毒素(hha)和细菌酸性调节因子(ari R) mRNA的表达量显著提高,抗毒素...     

在大肠杆菌中拓展合成途径合成异胡椒醇北大核心

天然异胡椒醇作为一种植物来源香料,具有很高的经济价值。目前国内外还未见利用大肠杆菌工程菌株转化生产异胡椒醇的报道。【目的】构建工程菌,采用生物合成方法获得天然香料异胡椒醇。【方法】对来自胡椒薄荷的细胞色素P450家族的柠檬烯-3-羟化酶(LIM3H)基因PM2进行改造,截短LIM3H的N端疏水区,分别添加17α短肽或2B1短肽增加其亲水性,采用CO差示光谱法检测LIM3H活性;将N端疏水区截短后的来自拟南芥(Arabidopsis thaliana)的NADPH-细胞色素P450还原酶基因(trATR)和修饰后LIM3H基因(Modi-LIM3H)融合后表达,最终以柠檬烯为底物进行全细胞转化。【结果】3种N端修饰LIM3H基因均检测到LIM3H表达和异胡椒醇生成,其中17α短肽修饰后的蛋白表达量最高,异胡椒醇产量达到1.94 mg/L。【结论】本研究在大肠杆菌中增加外源LIM3H与ATR,成功催化柠檬烯生成异胡椒醇,为天然异胡椒醇的生产提供了新的方法。     

The Effect of Ser 128 Substitution on the Structure and Stability of cAMP Receptor Protein from Escherichia coli

Kinetic measurements of denaturation and renaturation of two mutants of cAMP receptor protein (CRP) at position 128, namely SerAla and SerPro, were performed in order to assess changes introduced by the mutation in the quaternary structure and protein stability. No significant changes were found in the unfolding/refolding reactions. However, small perturbations in the dissociation of CRP dimer can be seen, which indicate that subunit interactions are influenced by the mutation. Studies of intrinsic fluorescence quenching of these two mutants are also reported, showing changes compared with wild-type protein. Near-UV circular dichroism measurements indicate, however, that Trp residues remain in the same environment as in the wild-type CRP. It is proposed that Ser at position 128 is involved in maintaining the proper domain alignment within CRP subunits.     

Cloning and sequencing of the HU-2 gene of Escherichia coli

Summary The Escherichia coli HU-2 gene was cloned using a DNA fragment from the HU-1 gene as a probe. The amino acid sequence of the HU-2 protein deduced from the nucleotide sequence is in good agreement with the published sequence. The nucleotide sequence has a possible promoter and a typical ribosomal binding site upstream of the translation initiation codon (AUG) and a possible rhoindependent terminater site downstream of the termination codon (UAA) of the gene.     

Properties of RecA441 protein reveal a possible role for RecF and SSB proteins in Escherichia coli

Summary We examined the possibility that the recA441 mutation, which partially suppresses the UV sensitivity of uvr recF mutant bacteria, exerts its effect by coding for an altered RecA protein that competes more efficiently than the RecA⁺ protein with SSB for ssDNA in vivo. Using an assay measuring recombination between UV-damaged DNA and intact homologous DNA, we found that the introduction of the recA441 mutation partially suppressed the defects in recombination in bacteria lacking RecF activity but not in bacteria with excess SSB, although recombination was affected more in recF mutants than in bacteria overproducing SSB. These results therefore do not support the hypothesis that RecA441 protein, or RecA protein with the help of RecF protein, is required during recombination of UV-damaged DNA to compete with SSB for ssDNA.     

米曲霉（Aspergillus oryzae）果胶酸内切水解酶（PGA）在原核系统中重组表达

果胶酶具有广阔的商业用途,在食品工业上主要用于果汁和酒类的澄清、提高植物油的提取率、提高水果的硬度和植物纤维脱胶。米曲霉（Aspergillus oryzae）一直用于传统发酵食品的生产,自然条件下其果胶酶的产量较低。文献报道的果胶酶的重组表达成功的例子较少,且活性较低。通过RT-PCR 的方法,获得不含信号肽的果胶酸内切水解酶A(polygalacturonase A, PGA) 的cDNA,PGA cDNA 连入pET-28a (+)载体, 构建 pET-28a (+)-pga 质粒。pET-28a (+)-pga 转化Turner (DE3) placⅠ细胞,得到转化子pET-28a (+)-pga-Turner (DE3) placⅠ,首次实现了米曲霉PGA在大肠杆菌系统中过表达,进一步对PGA在大肠杆菌系统中表达的条件进行了研究。在37℃、220 r/min条件培养pET-28a (+)-pga-Turner (DE3) placⅠ细胞,OD₆₀₀至 0.8左右时,用500μmol/L isopropyl β-D-thiogalactogalactopyranoside (IPTG)进行诱导表达,在15℃和170r/min条件下继续培养24 h,表达效果最好,相对于每毫升培养基而言,产酶可达到70u/mL,是米曲霉自然条件产酶量的87.5倍,远优于文献报道的重组表达的PGA酶活     

米曲霉（Aspergillus oryzae）果胶酸内切水解酶（PGA）在原核系统中重组表达

果胶酶具有广阔的商业用途,在食品工业上主要用于果汁和酒类的澄清、提高植物油的提取率、提高水果的硬度和植物纤维脱胶。米曲霉（Aspergillus oryzae）一直用于传统发酵食品的生产,自然条件下其果胶酶的产量较低。文献报道的果胶酶的重组表达成功的例子较少,且活性较低。通过RT-PCR 的方法,获得不含信号肽的果胶酸内切水解酶A(polygalacturonase A, PGA) 的cDNA,PGA cDNA 连入pET-28a (+)载体, 构建 pET-28a (+)-pga 质粒。pET-28a (+)-pga 转化Turner (DE3) placⅠ细胞,得到转化子pET-28a (+)-pga-Turner (DE3) placⅠ,首次实现了米曲霉PGA在大肠杆菌系统中过表达,进一步对PGA在大肠杆菌系统中表达的条件进行了研究。在37℃、220 r/min条件培养pET-28a (+)-pga-Turner (DE3) placⅠ细胞,OD₆₀₀至 0.8左右时,用500μmol/L isopropyl β-D-thiogalactogalactopyranoside (IPTG)进行诱导表达,在15℃和170r/min条件下继续培养24 h,表达效果最好,相对于每毫升培养基而言,产酶可达到70u/mL,是米曲霉自然条件产酶量的87.5倍,远优于文献报道的重组表达的PGA酶活     

重组蛋白融合信号肽在大肠杆菌周质空间表达的研究进展

重组蛋白在大肠杆菌中表达时,往往面临着形成包涵体的问题,而重组蛋白若是分泌至周质空间则基本解决了这一问题,周质空间的周质蛋白不仅能帮助重组蛋白正确折叠还有利于二硫键的生成。信号肽是一段由15-30个氨基酸组成,被融合在重组蛋白N端的短肽,按照结构、功能的不同可以划分为N区、H区和C区,具有引导重组蛋白转运至细胞周质空间的作用。本文综述了信号肽的结构组成、作用机理和基本分泌途径,讨论了信号肽的高效转运和筛选方法,总结了在大肠杆菌中重组蛋白融合信号肽实现周质表达的新进展,并对未来高效信号肽选择方面的研究进行了探讨。     

Recombinant streptokinase production by fed-batch cultivation of Escherichia coli

Streptokinase (SK) is a specific effective medicine for thrombolytic therapy of acute myocardial infarction. This study established a process for the pilot production of recombinant streptokinase (r-SK). Engineering bacteria were fermented in a 20-l fermentor to produce r-SK. After simple renaturation and purification, 12.9 g of r-SK with 97.8% of purity and about 10⁵ IU mg⁻¹ of specific activity was obtained, the yield of protein and the recovery of activity were 44.9% and 51%, respectively. Finally, the r-SK was made into about 700 doses of injections for clinical applications.     

大肠杆菌中转录-翻译耦合的机制

在大肠杆菌（Escherichia coli,E. coli）等原核生物中,转录和翻译往往是耦合的,这种耦合通常表现在转录和翻译的互相调控上,如转录极性、转录衰减和转录-翻译速率的同步。间接耦合和物理耦合是耦合的两种模式。由警报素（alarmone）(p)ppGpp维持的间接耦合可能需要DksA和TufA蛋白的辅助。物理耦合分为NusG或RfaH因子介导的耦合和非因子条件下产生的“碰撞”耦合。响应于压力的转录或翻译的变化会引发几种耦合模式间的相互转变。耦合对于基因正常表达是必要的,其解除将引发转录终止、R环形成、复制-转录冲突、mRNA切割等不利的事件。结构生物学的相关技术已经清晰地展示了部分耦合的表达体（expressome）的结构细节和特征,这些技术联合多组学分析等方法将提供关于耦合的更深层次的见解。重要的是,对耦合的研究或许会为靶向抗菌药物的开发带来新的思路。     

大肠杆菌胞内半胱氨酸响应型启动子的筛选表征

【目的】半胱氨酸是一种重要的含硫氨基酸,广泛应用于化妆品、药品、食品等行业,微生物发酵法合成半胱氨酸已成为当前研究的热点。基于比较转录组学分析等技术,筛选并表征大肠杆菌(Escherichia coli)胞内对半胱氨酸浓度变化显著响应的启动子。【方法】在Escherichia coli W3110培养基中外源添加不同终浓度的半胱氨酸,通过比较转录组学分析筛选转录水平显著响应半胱氨酸浓度变化的基因,融合目标基因的启动子片段与荧光报告基因egfp构建启动子文库,进一步测定不同半胱氨酸浓度条件下,含有不同启动子重组菌的绿色荧光蛋白(greenfluorescent protein, GFP)荧光强度。【结果】筛选并挖掘了随着半胱氨酸浓度提高而转录水平显著提升的27个基因,并将基因的潜在启动子片段与荧光报告基因egfp融合构建启动子文库,筛选获得对半胱氨酸变化具备特异性响应的启动子P_E2。最后,对P_E2启动子-35区间隔区域AAAT进行随机突变,最终获得在1-7g/L半胱氨酸浓度范围内特异性响应性能显著提高的启动子P_E2-33。...     

合成生物学方法构建电活性大肠杆菌

电活性微生物具有独特的在细胞内外环境之间传递电子的能力。在对天然电活性微生物电子传递机制充分研究的基础上,通过合成生物学方法异源构建天然电活性微生物电子传递结构基础也可以将遗传背景清晰的非电活性大肠杆菌改造为电活性微生物。构建获得的工程化电活性大肠杆菌可以直接应用于微生物燃料电池和生物传感器等领域,同时也可以作为底盘细胞整合相应的目标产物合成通路实现电能驱动的生物合成。本文以合成生物学方法构建电活性大肠杆菌为主题,详细阐述天然电活性微生物电子传递的机理及结构基础,总结了工程化电活性大肠杆菌的构建策略、成功案例以及应用领域,并对合成生物学方法构建电活性大肠杆菌未来的研究方向进行了展望。     

c-di-GMP对大肠杆菌生物膜调控的研究进展

大肠杆菌生物膜是由聚集于特定介质上的大肠杆菌菌体细胞相互黏附并分泌胞外基质聚合物(extracellular polymeric substances,EPS)而产生的一种结构复杂的膜状聚集物。感染宿主后的致病性大肠杆菌在形成生物膜后会极大地逃避免疫系统以及环境中各种有害因素对其的影响,对宿主造成持续甚至致命的伤害。环二鸟苷酸(cyclic diguanosine monophosphate,c-di-GMP)是广泛存在于细菌中的第二信使,在调节生物膜形成过程中起到至关重要的作用。基于此,本文对近些年来有关c-di-GMP对大肠杆菌生物膜形成过程中菌体的运动、黏附以及EPS产生机制的研究进行了综述,以期为从c-di-GMP角度抑制大肠杆菌生物膜提供依据和思路。     

The role of the carboxy-terminal membrane-spanning fragment in the biogenesis of Escherichia coli K12 outer membrane protein PhoE

Summary PhoE protein of Escherichia coli K12 is an outer membrane protein which is supposed to span the membrane sixteen times. By creating a deletion which removes the last membrane-spanning fragment and studying the localization of the truncated PhoE, we show that this fragment is indispensable for trimerization and outer membrane localization. In addition, circumstantial evidence for the proposed topology model of the protein was obtained. An insertion mutation in a region supposed to be cell surface-exposed, interferes with the binding of a monoclonal antibody which recognizes a cell surface-exposed epitope of the protein.     

产麦角硫因大肠杆菌工程菌株的构建与优化

麦角硫因(ergothioneine,ERG)是一种天然的抗氧化剂,广泛应用于化妆品、食品以及医药领域.相比于传统植物提取和化学合成方法,微生物发酵合成麦角硫因具有周期短、成本低等优点,因而受到广泛关注.为构建高产麦角硫因的大肠杆菌工程菌株,本研究以大肠杆菌(Escherichia coli) BL21 (DE3)为出...     

鸡源大肠杆菌毒力基因检测及分子流行特征

[目的] 本试验旨在阐明鸡源大肠杆菌致病性及分子流行特性,为探索大肠杆菌流行途径制定合理的防控策略提供新思路。[方法] 2018–2019年在河北省采集病死鸡肝脏样品,通过选择培养基筛选、生化鉴定、血清凝集试验对分离菌株进行系统鉴定,应用PCR方法检测分离株中毒力基因流行情况。参考系统发育群分类方法对大肠杆菌进行分群分析,并参照McMLST网站数据库提供的7对管家基因序列进行多位点序列分型（multilocus sequence typing,MLST）分析。[结果] 结果显示,56株分离株符合大肠杆菌生化特征,分为8个生化表型,B4（30.36%）、B5（25%）和B2（23.21%）为主要生化表型。56株分离株大肠杆菌血清凝集试验均呈阳性,分为11种血清型,O₇₈（26.79%）、O₂（23.21%）、O₁₅₇（17.86%）和O₁（14.29%）为主要流行血清型。56株大肠杆菌共检测出15种肠外大肠杆菌毒力基因,未检出papC、ibeA和ibeB基因。黏附相关基因fimC和抗血清存活因子相关基因ompA携带率为100%。aatA、yijP、irp2、mat、iss,检出率分别为98.21%、98.21%、98.21%、96.43%、92.86%。同时,大肠杆菌与铁转运相关基因iroN、fyuA、iucD、irp2检出率均在80%以上。56株大肠杆菌中有20株属于肠出血型大肠杆菌（enterohemorrhagic E.coli,EHEC）,其次是肠聚集型大肠杆菌（enteroaggregative E.coli,EAEC）（n=4）、肠产毒素型大肠杆菌（Enterotoxigenic E.coli,ETEC）（n=2）。这些菌株D群分离株较多,其次是B2群。通过MLST分型分析,共分为22个ST型,其中ST88（n=7）、ST85（n=6）、ST243（n=6）型为主要流行型。[结论] 结果显示大肠杆菌血清型多样,毒力因子种类繁多,致病性大肠杆菌同时携带多种毒力基因,表明动物源大肠杆菌具有较强的毒力基础。     

Escherichia coli expression and in vitro activation of a unique ligninolytic peroxidase that has a catalytic tyrosine residue

Heterologous expression of Trametes cervina lignin peroxidase (LiP), the only basidiomycete peroxidase that has a catalytic tyrosine, was investigated. The mature LiP cDNA was cloned into the pET vector and used to transform Escherichia coli. Recombinant LiP protein accumulated in inclusion bodies as an inactive form. Refolding conditions for its in vitro activation—including incorporation of heme and structural Ca²⁺ ions, and formation of disulfide bridges—were optimized taking as a starting point those reported for other plant and fungal peroxidases. The absorption spectrum of the refolded enzyme was identical to that of wild LiP from T. cervina suggesting that it was properly folded. The enzyme was able to oxidize 1,4-dimethoxybenzene and ferrocytochrome c confirming its high redox potential and ability to oxidize large substrates. However, during oxidation of veratryl alcohol (VA), the physiological LiP substrate, an unexpected initial lag period was observed. Possible modification of the enzyme was investigated by incubating it with H₂O₂ and VA (for 30 min before dialysis). The pretreated enzyme showed normal kinetics traces for VA oxidation, without the initial lag previously observed. Steady-state kinetics of the pretreated LiP were almost the same as the recombinant enzyme before the pretreatment. Moreover, the catalytic constant (k_cat) for VA oxidation was comparable to that of wild LiP from T. cervina, although the Michaelis–Menten constant (K_m) was 8-fold higher. The present heterologous expression system provides a valuable tool to investigate structure–function relationships, and autocatalytic activation of the unique T. cervina LiP.     

Resistance to trifluoroperazine, a calmodulin inhibitor, maps to the fabD locus in Escherichia coli

A mutant, tfpA1, resistant to the calmodulin inhibitor trifluoroperazine (TFP) at 30°C, was isolated in Escherichia coli. The mutant showed a reduced growth rate at 30°C and was temperature sensitive (ts) at 42°C for growth, forming short filaments. The mutation was mapped to the 24 min region of the chromosome and the gene was cloned by complementation of the is defect. Subsequent subcloning, complementation analysis, marker rescue mapping and sequencing, identified tfpA as fabD, encoding the 35 kDa, malonyl-coenzyme A transacylase (MCT) enzyme, required for the initial step in the elongation cycle for fatty acid biosynthesis. Resistance to TFP may result from altered permeability of the cell envelope, although the mutant remained sensitive to other calmodulin inhibitors and to other antibacterial agents. Alternatively, resistance may be more indirect, resulting from alterations in intracellular Ca⁺⁺ levels which affect the activity of the TFP target in some way.