大肠杆菌海藻糖的代谢调控

海藻糖是一种重要的抗逆物质。大肠杆菌中otsBA操纵子编码的两种酶负责海藻糖合成。otsBA基因的表达受渗透压诱导和σs因子的调节。细胞的周质海藻糖酶(treA)将外源海藻糖分解成两个葡萄糖分子。尽管大肠杆菌中渗透压诱导合成的海藻糖并不能保护细胞抗干燥,我们将otsA单个基因通过农杆菌转入烟草时,转基因株提高了耐盐和抗干燥特性,同时在转基因烟草提取物中检测到海藻糖,证明otsA基因在烟草中表达并合成海藻糖。我们认为若将otsA基因转入其它植物,可望改善这些植物的抗干旱、耐盐碱特性和延长采摘后的保鲜期 。     

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。     

大肠杆菌海藻糖的代谢调控

海藻糖是一种重要的抗逆物质。大肠杆菌中ｏｔｓＢＡ操纵子编码的两种酶负责海藻糖合成。ｏｔｓＢＡ基因的表达受渗透压诱导和σ＾ｓ因子的调节。细胞的周质海藻糖酶（ｔｒｅＡ）将外源海藻藻分解成两个葡萄糖分子。尽管大肠杆菌中渗透压诱导合成的海藻糖并不能保护细胞抗干燥,我们将ｏｔｓＡ单个基因通过农杆菌转入烟草时,转基因株提高了耐盐和抗干燥特性,同时在转基因烟草提取物中检测到海藻糖,证明ｏｔｓＡ基因在烟草中表达并合成海藻糖。我们认为若将ｏｔｓＡ基因转入其它植物,可望改善这些植物的抗干旱、耐盐碱特性和延长采摘后的保鲜期。     

外源蛋白(BglS)在大肠杆菌中的合成、定域和加工

B.subtilis中编码β-1,3-1,4葡聚糖酶的基因bglS,它的产物在E.coli中的合成受宿主、载体和基因片段插入方向的影响。从E.coli细胞中BglS蛋白质定域分析和SDS-PAGE中活性酶分子检测发现存在着32kD和27kD两种活性酶分子,酶分子在周质空间很少停留。这种不同的活性酶分子的出现和分泌特点,可能与大肠杆菌细胞蛋白质加工和转位的方式有关。     

信号肽在大肠杆菌分泌系统中的研究与应用进展

大肠杆菌以其明显的优势成为表达重组蛋白常用的系统,但是大肠杆菌本身不具备细胞内形成二硫键的氧化条件和分子机制,而且高水平表达时常容易聚集形成包涵体,限制了其使用,改善这一缺点的重要方法是通过信号肽实现蛋白质的分泌表达。信号肽一般存在于分泌蛋白的氨基端,能够引导蛋白质通过大肠杆菌中的Sec或/和Tat系统分泌至周质空间。简要概述了大肠杆菌中两种跨膜分泌系统和信号肽的结构,并结合近年来常用6种信号肽的研究与应用进展,阐述了信号肽在使用中存在的问题及改进措施。旨在为研究者合理选择信号肽、优化重组蛋白的表达提供更多可用的信息与策略。     

奥奈达希瓦氏菌CctA介导周质甲基橙还原的电子传递机理

电活性微生物奥奈达希瓦氏菌的胞外电子传递（extracellular electron transfer,EET）在污染物降解、环境修复、生物电化学传感、能源利用等方面具有广泛的应用潜力;四血红素细胞色素CctA （small tetraheme cytochrome）是希瓦氏菌周质空间中最丰富的蛋白质之一,能够参与多种氧化还原过程,但目前对CctA在EET中的行为和机理认识仍然有限。【目的】研究阐明CctA蛋白在希瓦氏菌模式菌株MR-1周质空间以偶氮染料作为电子受体的EET中的作用,补充和拓展希瓦氏菌的厌氧呼吸产能机制。【方法】以周质还原型偶氮染料甲基橙（methyl orange,MO）作为电子受体,在mteal reduction （Mtr）蛋白缺失菌株Δmtr中研究MO的周质还原特点,并通过基因敲除和回补表达研究CctA蛋白在周质电子传递中的作用。【结果】在缺失Mtr通道的情况下,细胞色素CctA可以介导周质空间的电子传递而还原MO。重组表达CctA在低水平时,MO在周质空间中的还原速率与其表达水平呈正相关,更高水平的CctA表达无助于进一步提高MO的还原速率。蛋白膜伏安结果展示了CctA与周质空间内其他高电位氧化还原蛋白的显著区别,可能参与构成一条低电位的MO还原通道。【结论】从分子动力学层面揭示了CctA在周质MO还原中的独特电子传递行为,为进一步推进对细菌周质电子传递机制的理解,以及通过合成生物学设计或改造胞外氧化还原系统、强化生物电化学在污染物降解中的应用提供了重要信息。     

多聚磷酸盐在微生物抗环境胁迫中的作用及机制

抗环境胁迫是微生物提高环境适应性和增加生存机会的一个重要策略，探明微生物抗环境胁迫的过程及分子机制对于了解微生物进化和开发微生物资源具有重要意义。多聚磷酸盐（polyphosphate, polyP）在微生物抗环境胁迫中发挥重要作用。在营养限制条件下，polyP可充当微生物的能源来源和信号分子，增强微生物对低营养环境的适应能力。在微生物应对环境胁迫过程中，polyP可作为蛋白质的伴侣，通过蛋白质修饰改变蛋白质结构使其免受失活，从而维持其功能完整性。polyP具有金属螯合能力，可提高微生物对重金属胁迫的抵抗能力。微生物能通过调节polyP的合成来适应环境pH的改变，调节酸碱胁迫过程中的能量消耗。基于polyP抗环境胁迫的特性，通过转基因技术，把polyP合成相关基因转入到农作物中，可以增加农作物体内polyP含量，从而提高农作物抗环境胁迫的能力。利用含有polyP的微生物处理重金属废水，可极大地提高重金属离子的去除效率。同时，微生物中合成的polyP颗粒也能进一步开发为生物活性产品。因此，polyP在微生物抗胁迫中发挥多样化作用，通过各种分子途径提高微生物对环境胁迫的耐受性。加强poly...     

大肠杆菌的蛋白分泌机制

大肠杆菌的分泌蛋白定位于内膜、外膜、周质空间和胞外环境,它们在N端或C端带有一定的结构包含着分泌信号,这两类分泌蛋白在各自特定的一组蛋白因子的协助下跨越内膜,再通过目前尚不清楚的方式实现其最终定位.N端带有信号肽的分子在跨越内膜时得到Sec家族蛋白因子协助,信号肽在跨膜过程中可能被切除,该过程由ATP和电化学势提供能量.C端带分泌信号的分子主要受到Hly家族分子协助,一次穿过内膜和外膜而不经过周质空间.     

蛋白质在大肠杆菌间周质中的折叠

随着分子伴侣的发现和外源基因表达技术的发展 ,大肠杆菌间周质蛋白质的折叠成为研究热点。间周质 (ｐｅｒｉｐｌａｓｍ)是革兰氏阴性菌内膜与外膜之间的区域 ,对外界环境变化的适当能力很脆弱 ,例如ｐＨ、温度、渗透压[1] 。重组技术表达的重要蛋白质大多含有二硫键 ,二硫键的形成在真核生物是在内质网中完成的 ,而大肠杆菌是在间周质中进行的。催化大肠杆菌间周质蛋白质折叠会遇到两个限速步骤 ,被两类酶催化 :二硫键氧化还原酶 (Ｄｓｂ)和肽酰 脯氨酰顺反异构酶 (ｐｅｐｔｉｄｙｌ ｐｒｏｌｙｌｃｉｓ ｔｒａｎｓｉｓｏ ｍｅｒａｓｅ ,…     

Kil蛋白介导的大肠杆菌外泌表达系统

利用大肠杆菌素释放基因（kil)能有效地增加细菌外膜通透性促进周质蛋白外源的原理构建了含kil基因的大肠杆菌外泌表达系统,将大肠杆菌本身的周质分泌蛋白β-内酰胺酶和异源周质分泌蛋白点状产气单胞菌脯氨酰内肽酶作为报告蛋白,观察Kil蛋白对这两种通常极少分泌到胞外的周质蛋白胞外分泌的促进作用,我们的研究显示：kil基因表达时,β-内酰胺酶的总活性较对照组提高1倍,胞外分泌活性较对照组提高近4倍,脯氨酰内肽酶的总活性较对照组提高0.8倍,胞外分泌活性较对照组提高3倍。     

pH-dependent expression of periplasmic proteins and amino acid catabolism in Escherichia coli

Escherichia coli grows over a wide range of pHs (pH 4.4 to 9.2), and its own metabolism shifts the external pH toward either extreme, depending on available nutrients and electron acceptors. Responses to pH values across the growth range were examined through two-dimensional electrophoresis (2-D gels) of the proteome and through lac gene fusions. Strain W3110 was grown to early log phase in complex broth buffered at pH 4.9, 6.0, 8.0, or 9.1. 2-D gel analysis revealed the pH dependence of 19 proteins not previously known to be pH dependent. At low pH, several acetate-induced proteins were elevated (LuxS, Tpx, and YfiD), whereas acetate-repressed proteins were lowered (Pta, TnaA, DksA, AroK, and MalE). These responses could be mediated by the reuptake of acetate driven by changes in pH. The amplified proton gradient could also be responsible for the acid induction of the tricarboxylic acid (TCA) enzymes SucB and SucC. In addition to the autoinducer LuxS, low pH induced another potential autoinducer component, the LuxH homolog RibB. pH modulated the expression of several periplasmic and outer membrane proteins: acid induced YcdO and YdiY; base induced OmpA, MalE, and YceI; and either acid or base induced OmpX relative to pH 7. Two pH-dependent periplasmic proteins were redox modulators: Tpx (acid-induced) and DsbA (base-induced). The locus alx, induced in extreme base, was identified as ygjT, whose product is a putative membrane-bound redox modulator. The cytoplasmic superoxide stress protein SodB was induced by acid, possibly in response to increased iron solubility. High pH induced amino acid metabolic enzymes (TnaA and CysK) as well as lac fusions to the genes encoding AstD and GabT. These enzymes participate in arginine and glutamate catabolic pathways that channel carbon into acids instead of producing alkaline amines. Overall, these data are consistent with a model in which E. coli modulates multiple transporters and pathways of amino acid consumption so as to minimize the shift of its external pH toward either acidic or alkaline extreme.     

HDEA, a periplasmic protein that supports acid resistance in pathogenic enteric bacteria

The X-ray crystal structure of the Escherichia coli stress response protein HDEA has been determined at 2.0 A resolution. The single domain alpha-helical protein is found in the periplasmic space, where it supports an acid resistance phenotype essential for infectivity of enteric bacterial pathogens, such as Shigella and E. coli. Functional studies demonstrate that HDEA is activated by a dimer-to-monomer transition at acidic pH, leading to suppression of aggregation by acid-denatured proteins. We suggest that HDEA may support chaperone-like functions during the extremely acidic conditions.     

Expression and Export of Pseudomonas putida NTU-8 Creatinase by Escherichia coli Using the Chitinase Signal Sequence of Aeromonas hydrophila

The gene for the creatinase from Pseudomonas putida NTU-8 was sequenced and revealed an open reading frame (ORF) of 1209 base pairs encoding a polypeptide of 403 amino acids with a calculated molecular weight (M(r)) of 45,691. The deduced amino acid sequence is very similar to that of the creatinase of Pseudomonas putida and Flavobacterium sp. An overproduction system for the chitinase signal peptide--creatinase hybrid gene was constructed by using the pQE-51 expression vector in E. coli JM109. The amount of this fusion enzyme was about 50% exported into the periplasmic space of E. coli.     

Products of the Escherichia coli acid fitness island attenuate metabolite stress at extremely low pH and mediate a cell density-dependent acid resistance

Escherichia coli has an ability, rare among the Enterobacteriaceae, to survive extreme acid stress under various host (e.g., human stomach) and nonhost (e.g., apple cider) conditions. Previous microarray studies have exposed a cluster of 12 genes at 79 centisomes collectively called an acid fitness island (AFI). Four AFI genes, gadA, gadX, gadW, and gadE, were already known to be involved in an acid resistance system that consumes an intracellular proton through the decarboxylation of glutamic acid. However, roles for the other eight AFI gene products were either unknown or subject to conflicting findings. Two new aspects of acid resistance are described that require participation of five of the remaining eight AFI genes. YhiF (a putative regulatory protein), lipoprotein Slp, and the periplasmic chaperone HdeA protected E. coli from organic acid metabolites produced during fermentation once the external pH was reduced to pH 2.5. HdeA appears to handle protein damage caused when protonated organic acids diffuse into the cell and dissociate, thereby decreasing internal pH. In contrast, YhiF- and Slp-dependent systems appear to counter the effects of the organic acids themselves, specifically succinate, lactate, and formate, but not acetate. A second phenomenon was defined by two other AFI genes, yhiD and hdeD, encoding putative membrane proteins. These proteins participate in an acid resistance mechanism exhibited only at high cell densities (>10(8) CFU per ml). Density-dependent acid resistance does not require any demonstrable secreted factor and may involve cell contact-dependent activation. These findings further define the complex physiology of E. coli acid resistance.     

Acinetobacter calcoaceticus encoded mutarotase: nucleotide sequence analysis of the gene and characterization of its secretion in Escherichia coli.

The nucleotide sequence of the mutarotase gene from Acinetobacter calcoaceticus has been determined. It reveals an open reading frame of 381 amino acids. The codon usage of A. calcoaceticus for this gene is similar to E. coli except for the amino acids Leu, Ala, Glu, and Arg where major differences exist. This did not interfere drastically with high level expression in E. coli. The regulatory sequences for the initiation of translation are similar to the ones described for E. coli. The N-terminal 20 amino acids, which are not found in the mature enzyme, show homology to signal sequences of exported proteins. In A. calcoaceticus and E. coli mutarotase is specifically secreted into the periplasmic space. Processing of the signal sequence occurs at identical sites in both organisms. The mature mutarotase consists of 361 amino acids and has a calculated molecular weight of 38457 Da. Expression of mutarotase at a high level in a recombinant E. coli destabilizes the outer membrane. This results in coordinated leakage of mutarotase and beta-lactamase into the culture broth.     

Secretion activities of Bacillus subtilis alpha-amylase signal peptides of different lengths in Escherichia coli cells

The B. subtilis alpha-amylase promoter and signal peptide are functional in E. coli cells. DNA fragments coding for signal peptides with different lengths (28, 31, 33 and 41 amino acids from the translation initiator Met) were prepared and fused with the E. coli beta-lactamase structural gene. In B. subtilis cells, the sequences of 31, 33 and 41 amino acids were able to secrete beta-lactamase into the surrounding media, but the 28 amino acid sequence was not. In contrast, all of the four sequences were able to export beta-lactamase into the periplasmic space of E. coli cells. Thus, the recognition of the B. subtilis alpha-amylase signal peptide in E. coli cells seems to be different from that in B. subtilis cells.     

alpha-Amylase of Clostridium thermosulfurogenes EM1: nucleotide sequence of the gene, processing of the enzyme, and comparison of other alpha-amylases

The nucleotide sequence of the alpha-amylase gene (amyA) from Clostridium thermosulfurogenes EM1 cloned in Escherichia coli was determined. The reading frame of the gene consisted of 2,121 bp. Comparison of the DNA sequence data with the amino acid sequence of the N terminus of the purified secreted protein of C. thermosulfurogenes EM1 suggested that the alpha-amylase is translated from mRNA as a secretory precursor with a signal peptide of 27 amino acid residues. The deduced amino acid sequence of the mature alpha-amylase contained 679 residues, resulting in a protein with a molecular mass of 75,112 Da. In E. coli the enzyme was transported to the periplasmic space and the signal peptide was cleaved at exactly the same site between two alanine residues. Comparison of the amino acid sequence of the C. thermosulfurogenes EM1 alpha-amylase with those from other bacterial and eucaryotic alpha-amylases showed several homologous regions, probably in the enzymatically functioning regions. The tentative Ca(2+)-binding site (consensus region I) of this Ca(2+)-independent enzyme showed only limited homology. The deduced amino acid sequence of a second obviously truncated open reading frame showed significant homology to the malG gene product of E. coli. Comparison of the alpha-amylase gene region of C. thermosulfurogenes EM1 (DSM3896) with the beta-amylase gene region of C. thermosulfurogenes (ATCC 33743) indicated that both genes have been exchanged with each other at identical sites in the chromosomes of these strains.