Primary structure of the CFA1 fimbrial protein from human enterotoxigenic Escherichia coli strains

The complete amino acid sequence of the structural protein that constitutes the subunit of the CFA1 fimbria has been elucidated. The protein was fragmented by cyanogen bromide cleavage, and by enzymatic cleavage with trypsin. Secondary cleavage of the resulting peptides was performed with chymotrypsin, Staphylococcus aureus protease, and thermolysin. Sequential Edman degradation was performed manually. The CFA1 protein comprises 147 amino acid residues, with a molecular weight of 15058.     

A novel inhibitor protein of N-myristoyltransferase from Escherichia coli

Myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyzes the covalent attachment of myristate to the N-terminal of the glycine residue of various eukaryotic and viral proteins of diverse functions. Earlier, we have demonstrated that NMT activity is elevated in colon and gall bladder cancer. Attenuation of NMT activity may prove a novel therapeutic protocol for cancer. We report here a novel inhibitor protein of NMT being expressed in Escherichia coli cells containing the human NMT gene on increasing the incubation period from 5 to 24h. The inhibitor protein was purified by SP-Sepharose column chromatography, heat treatment, ammonium sulfate precipitation, and Superose 12 HR/30 FPLC column chromatography. The inhibitor protein had an apparent molecular mass of 10kDa by gel filtration. It inhibited human NMT in a concentration-dependent manner with 50% inhibition at 640+/-4.68nM. The inhibitor protein showed no direct interaction with myristoyl-CoA and demonstrated no demyristoylase or protease activity. Therefore, we conclude that the inhibitor protein acts directly on NMT.     

Molecular serogrouping of porcine enterotoxigenic Escherichia coli from Australia

Enterotoxigenic Escherichia coli (ETEC) is a common etiological agent of neonatal, pre and post weaning diarrhoea in piglets. One of the most important steps in the diagnosis and epidemiological understanding of this organism is accurate serogrouping. In many instances, however, conventional serogrouping fails to produce accurate identification of serogroups. In this communication we report a modified and simplified molecular serogrouping method (rfb-RFLP) for the accurate identification of the most common porcine ETEC strains that cause neonatal, pre and post weaning diarrhoea in Australia.     

大肠杆菌K99菌毛fan操纵子的克隆、表达及活性

[目的]在体外克隆和表达猪产肠毒素大肠杆菌(ETEC) K99菌毛操纵子fan结构基因,并检测重组菌毛的相关生物学活性.[方法]以猪源分离的表达K99菌毛ETEC C83907株制取模板,成功PCR扩增出编码K99菌毛的fan操纵子,约5.7 kb.将fan操纵子克隆人表达质粒载体pBR322,筛选出含正确阳性质粒的重组菌.进一步将上述的重组质粒DNA转化至不含任何菌毛的大肠杆菌SE5000株,同时将空载体pBR322质粒转化入SE5000构建阴性对照菌株.[结果]该重组菌能与鼠抗K99菌毛单克隆抗体发生明显的凝集反应,与新生仔猪小肠上皮细胞刷状缘BBV分子有强烈凝集反应.电镜观察到上述重组菌表面大量表达K99菌毛,用热抽提法提纯其表达的K99菌毛,并经SDS-PAGE电泳和考马斯亮蓝染色,可以得到分子量约为18.5kDa的主要蛋白条带.纯化菌毛免疫小鼠后制备出高效价的鼠抗血清,能与携带K99菌毛的C83907、C83914、C83260野生株发生强烈的凝集反应,而与携带其他菌毛的ETEC不反应.玻板凝集试验和Western blot结果表明:体外表达的K99菌毛具有和野生K99菌毛相同的抗原性.用表达K99菌毛的重组菌进行HeLa细胞体外黏附试验和黏附抑制实验,结果表明:重组菌和野生菌株一样具有较强的粘附性,而且用重组菌毛制备的鼠抗血清能有效地抑制上述重组菌或野生菌株对细胞系的黏附结合.[讨论]本研究为进一步研究K99菌毛生物学作用建立了良好的实验平台.     

Induction and function of the phage shock protein extracytoplasmic stress response in Escherichia coli

The phage shock protein (Psp) F regulon response in Escherichia coli is thought to be induced by impaired inner membrane integrity and an associated decrease in proton motive force (pmf). Mechanisms by which the Psp system detects the stress signal and responds have so far remained undetermined. Here we demonstrate that PspA and PspG directly confront a variety of inducing stimuli by switching the cell to anaerobic respiration and fermentation and by down-regulating motility, thereby subtly adjusting and maintaining energy usage and pmf. Additionally, PspG controls iron usage. We show that the Psp-inducing protein IV secretin stress, in the absence of Psp proteins, decreases the pmf in an ArcB-dependent manner and that ArcB is required for amplifying and transducing the stress signal to the PspF regulon. The requirement of the ArcB signal transduction protein for induction of psp provides clear evidence for a direct link between the physiological redox state of the cell, the electron transport chain, and induction of the Psp response. Under normal growth conditions PspA and PspD control the level of activity of ArcB/ArcA system that senses the redox/metabolic state of the cell, whereas under stress conditions PspA, PspD, and PspG deliver their effector functions at least in part by activating ArcB/ArcA through positive feedback.     

Cnu, a novel oriC-binding protein of Escherichia coli

We have found, using a newly developed genetic method, a protein (named Cnu, for oriC-binding nucleoid-associated) that binds to a specific 26-base-pair sequence (named cnb) in the origin of replication of Escherichia coli, oriC. Cnu is composed of 71 amino acids (8.4 kDa) and shows extensive amino acid identity to a group of proteins belonging to the Hha/YmoA family. Cnu was previously discovered as a protein that, like Hha, complexes with H-NS in vitro. Our in vivo and in vitro assays confirm the results and further suggest that the complex formation with H-NS is involved in Cnu/Hha binding to cnb. Unlike the hns mutants, elimination of either the cnu or hha gene did not disturb the growth rate, origin content, and synchrony of DNA replication initiation of the mutants compared to the wild-type cells. However, the cnu hha double mutant was moderately reduced in origin content. The Cnu/Hha complex with H-NS thus could play a role in optimal activity of oriC.     

Structure-function analysis of the K88ab fimbrial subunit protein from porcine enterotoxigenic Escherichia coli

Several in-frame linker insertions have been made in various positions in the faeG gene encoding the K88ab fimbrial subunit protein from porcine enterotoxigenic Escherichia coli. The effects of the linker insertions have been investigated with regard to the ability of the mutated fimbrial subunits to be exported to the surface of the bacterial cell and assembled into a fimbrial structure. The structure/function relationship of the subunit protein is discussed in the light of the phenotypes of the mutations constructed, secondary structure predictions, and hydrophilicity plots.     

Charge heterogeneity of heat-labile enterotoxins from human enterotoxigenic Escherichia coli

Abstract We purified heat-labile enterotoxins (LThs) from YT3, H-10407 and YT240 strains isolated from human diarrheal patients. These LThs were immunologically identical to each other. The molecular weights of their A and B subunits were also the same by means of SDS-polyacrylamide gel electrophoresis. However, the ionic charges of the molecular surfaces of these LThs were different as shown by polyacrylamide gel isoelectric focusing. Though the p I points of B subunits of the LThs were identical to each other, the p I points of A subunits were found to be different. These data suggest that the ionic charge differences among A subunits cause differences in holo LThs in their charge, and that there is heterogeneity among A subunits produced by strains of human enterotoxigenic Escherichia coli .     

Molecular mechanisms of enterotoxigenic Escherichia coli infection

Enterotoxigenic Escherichia coli (ETEC) are a major cause of diarrheal illness in developing countries, and perennially the most common cause of traveller's diarrhea. ETEC constitute a diverse pathotype that elaborate heat-labile and/or heat-stable enterotoxins. Recent molecular pathogenesis studies reveal sophisticated pathogen–host interactions that might be exploited in efforts to prevent these important infections. While vaccine development for these important pathogens remains a formidable challenge, extensive efforts that attempt to exploit new genomic and proteomic technology platforms in discovery of novel targets are presently ongoing.     

Molecular cloning of actinohivin, a novel anti-HIV protein from an actinomycete, and its expression in Escherichia coli

Syncytium-inducing variants of the HIV-1 virus are correlated with poor diagnosis and rapid disease progression. We have recently discovered a novel anti-HIV protein, referred to as actinohivin, that inhibits syncytium formation. Here we describe the cloning and sequencing of the gene encoding actinohivin from the actinomycete strain K97-0003, and its expression in Escherichia coli. The actinohivin gene was located on a 0.8-kb BamHI fragment of genomic DNA. The fragment contained an open reading frame of 480 bp, which encoded a protein of 160 amino acids with calculated molecular weight of 17492.7. The N-terminal region was found to be a typical signal peptide of prokaryotes, and actinohivin was located at amino acid positions 46-160. The actinohivin gene could be expressed in E. coli using a pET30Xa/LIC expression vector and the purified recombinant actinohivin was found to inhibit syncytium formation to a similar extent as actinohivin from its natural source.     

Pathogenic effect of enterotoxigenic Escherichia coli and Escherichia coli causing infantile diarrhoea.

Macroscopic, light and electron microscopic alterations in ligated rabbit intestinal loops challenged with five standard enterotoxigenic Escherichia coli (ETEC) and twenty-three enteropathogenic E. coli (EEC-I) strains, freshly isolated from infantile enteritis cases, were investigated. Only two O26 : K60 : H11 strains produced enterotoxin. Their living cultures, sterile filtrates of the fluid medium and ultrasonic lysates of the bacteria resulted in pronounced hypersecretion of the intestinal epithelium followed by fluid accumulation and loop dilatation. These two E. coli strains, similarly as the other loop-negative EEC-I strains, were able to penetrate into the intestinal epithelium. In contrast to the standard ETEC strains, the EEC-I bacteria, adhering to the brush border, intruded into the microvilli, multiplied on the outer epithelial cell membrane making close contact with it and, causing, shedding of microvilli, penetrated into enterocytes becoming enclosed in membrane-bound phagosome-like vacuoles, appeared in the lamina propria and elicited mild focal polymorphonuclear infiltration.     

Recombinant expression and characterization of a novel endoglucanase from Bacillus subtilis in Escherichia coli

The goal of this work was to produce high levels of endoglucanase in Escherichia coli for its potential usage in different industrial applications. Endoglucanase gene was amplified from genomic DNA of Bacillus subtilis JS2004 by PCR. The isolated putative endoglucanase gene consisted of an open reading frame of 1,701 nucleotides and encoded a protein of 567 amino acids with a molecular mass of 63-kDa. The gene was cloned into pET-28a(+) and expressed in E. coli BL21 (DE3). Optimum temperature and pH of the recombinant endoglucanase were 50 °C and 9, respectively which makes it very attractive for using in bio-bleaching and pulp industry. It had a K _M of 1.76 μmol and V _max 0.20 μmol/min with carboxymethylcellulose as substrate. The activity of recombinant endoglucanse was enhanced by Mg²⁺, Ca²⁺, isopropanol and Tween 20 and inhibited by Hg²⁺, Zn²⁺, Cu²⁺, Ni²⁺ and SDS. The activity of this recombinant endoglucanase was significantly higher than wild type. Therefore, this recombinant enzyme has potential for many industrial applications involving biomass conversions, due to characteristic of broad pH and higher temperature stability.     

Development of a novel Gateway-based vector system for efficient, multiparallel protein expression in Escherichia coli

We describe a cloning and expression system which is based on the Escherichia coli T7 expression system and Gateway recombination technology. We have produced numerous destination vectors with selected fusion tags and an additional set of entry vectors containing the gene of interest and optional labeling tags. This powerful system enables us to transfer a cDNA to several expression vectors in parallel and combine them with various labeling tags. To remove the attached amino terminal tags along with the unwanted attB1 site, we inserted PreScission protease cleavage sites. In contrast to the commercially available destination vectors, our plasmids provide kanamycin resistance, which can be an advantage when expressing toxic proteins in E. coli. Some small-scale protein expression experiments are shown to demonstrate the usefulness of these novel Gateway vectors. In summary, this system has some benefits over the widely used and commercially available Gateway standard system, and it enables many different combinations for expression constructs from a single gene of interest.     

Crystal structure of enterotoxigenic Escherichia coli colonization factor CS6 reveals a novel type of functional assembly

Coli surface antigen 6 (CS6) is a widely expressed enterotoxigenic Escherichia coli (ETEC) colonization factor that mediates bacterial attachment to the small intestinal epithelium. CS6 is a polymer of two protein subunits CssA and CssB, which are secreted and assembled on the cell surface via the CssC/CssD chaperone usher (CU) pathway. Here, we present an atomic resolution model for the structure of CS6 based on the results of X‐ray crystallographic, spectroscopic and biochemical studies, and suggest a mechanism for CS6‐mediated adhesion. We show that the CssA and CssB subunits are assembled alternately in linear fibres by the principle of donor strand complementation. This type of fibre assembly is novel for CU assembled adhesins. We also show that both subunits in the fibre bind to receptors on epithelial cells, and that CssB, but not CssA, specifically recognizes the extracellular matrix protein fibronectin. Taken together, structural and functional results suggest that CS6 is an adhesive organelle of a novel type, a hetero‐polyadhesin that is capable of polyvalent attachment to different receptors.     

肠毒素源性定居因子CS6在减素伤寒沙门氏菌中表达及免疫原性的研究

将编码肠毒素源性大肠杆菌定居因子抗原ＣＳ６基因克隆到ｐＸＬ６７０,转化ａｓｄ基因突变的Ｅ．ｃｏｌｉ Ｘ６０９７,获得重组质粒ｐＳＳ６４,再将后者转化至减毒的△ａｒｏＡ、△ａｒｏＣ、△ａｓｄ伤寒沙门氏菌,构建了无药物抗性且稳定的大肠杆菌和伤寒双价菌苗候选株。小鼠腹腔免疫和攻击实验表明,该菌株对伤寒沙门氏菌毒株的攻击具有良好的保护作用。家兔免疫实验证明,该菌株能产生抗ＣＳ６和伤寒菌Ｖｉ抗原的血清抗体。     

An enhanced protocol for expression and purification of heat-stable enterotoxin of enterotoxigenic Escherichia coli

We present an improved protocol for expression and purification of heat-stable enterotoxin (STa) of enterotoxigenic Escherichia coli (ETEC). In this protocol, controlled growth conditions at different pHs (7.4, 8.0, and 8.6) were adopted using a bioreactor. In addition, specific adsorbent resins, methacrylate, were used for STa purification. The bioreactor provided optimal ETEC growth at pH 7.4 with high STa production. Furthermore, methacrylate bounded specifically to STa and dramatically enhanced the purification process of STa. The STa-specific activity was high (8.9 × 10(6) units/mg protein), and the minimal effective dose of STa required for production of gut weight to remaining body weight ratio ≥ 0.083 was recorded as less than 0.2 ng in 2-3 days old suckling mice. The protocol presented, produces highly purified STa as documented by matrix-assisted laser desorption ionization-time of flight mass spectroscopy/. Also, as compared with the traditional methods, this procedure is trouble-free and practical for scale-up production and purification of STa peptides.