Alkaline phosphatase isozyme conversion by cell-free extract of Escherichia coli

Isozyme type 1 of alkaline phosphatase in Escherichia coli K-12 was converted to types 2 and 3 after incubation of type 1 isozyme with the supernatant of a sonicated cell-free extract prepared from the cells carrying the cloned iap+ gene on a multi-copy plasmid. By comparison, the lysate prepared from cells carrying the iap+ gene only on the chromosome showed much less isozyme-converting activity. The reaction was promoted by Mg2+ at concentrations of 10 to 50 mM. Protease inhibitors, antipain and leupeptin which inhibit the isozyme conversion in vivo, also inhibited the isozyme conversion in vitro. These results suggest that cells carrying the multiple copy iap+ plasmid overproduce a kind of proteolytic enzyme which removes the amino-terminal arginine residues from isozymes 1 and 2.     

Characterisation of maltase from enteropathogenic Escherichia coli

Abstract Enteropathogenic strains of faecal Escherichia coli produced significantly ( P < 0.01) more maltase than the non-pathogenic strains of the organism. The enzyme was induced by maltose but repressed by glucose and fructose. The maltase was partially purified by ammonium sulphate precipitation, followed by dialysis and gel permeation chromatography. The partially purified maltase had an M _r of 144500 and an apparent K _m of approx. 7.6 mM for maltose. The enzyme was stimulated by Ca²⁺, inhibited by Cu²⁺, Hg²⁺, Uo²⁺, IAA and EDTA, and exhibited optimum activity at pH 6.5 at 30°C.     

The cytochromes of Escherichia coli

Abstract Escherichia coli contains numerous heme-containing proteins when grown either aerobicaly or anaerobically. These cytochrome species are distributed in the cytoplasm, the periplasm, or are bound to the cytoplasmic membrane. They are involved in various physiological functions, including electron transport, oxidative phosphorylation, assimilatory metabolism and detoxification. One dozen unique cytochrome species have been biochemically and/or genetically characterized. They contain one or more of the four heme groups which E. coli is known to produce: protoheme IX, heme c , heme d , and siroheme. The purpose of this articles is to summarize what we know about the structure and function of this collection of heme proteins.     

Impact of microbial diversity on rapid detection of enterohemorrhagic Escherichia coli in surface waters

Enterohemorrhagic Escherichia coli (EHEC) are a physiologically, immunologically and genetically diverse collection of strains that pose a serious water-borne threat to human health. Consequently, immunological and PCR assays have been developed for the rapid, sensitive detection of presumptive EHEC. However, the ability of these assays to consistently detect presumptive EHEC while excluding closely related non-EHEC strains has not been documented. We conducted a 30-month monitoring study of a major metropolitan watershed. Surface water samples were analyzed using an immunological assay for E. coli O157 (the predominant strain worldwide) and a multiplex PCR assay for the virulence genes stx(1), stx(2) and eae. The mean frequency of water samples positive for the presence of E. coli O157, stx(1) or stx(2) genes, or the eae gene was 50%, 26% and 96%, respectively. Quantitative analysis of selected enriched water samples indicated that even in samples positive for E. coli O157 cells, stx(1)/stx(2) genes, and the eae gene, the concentrations were rarely comparable. Seventeen E. coli O157 strains were isolated, however, none were EHEC. These data indicate the presence of multiple strains similar to EHEC but less pathogenic. These findings have important ramifications for the rapid detection of presumptive EHEC; namely, that current immunological or PCR assays cannot reliably identify water-borne EHEC strains.     

Shiga toxin-producing Escherichia coli and enteropathogenic Escherichia coli in healthy goats in India: occurrence and virulence properties

AIMS: To describe the occurrence and virulence gene pattern of shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) in healthy goats of Jammu and Kashmir, India. METHODS AND RESULTS: A total of 220 E. coli strains belonging to 60 different 'O' serogroups was isolated from 206 local (nonmigratory) and 69 migratory goats. All the 220 strains were screened for the presence of stx(1), stx(2), eaeA and hlyA genes. Twenty-eight E. coli (75.6%) strains from local and nine (24.3%) strains from migratory goats belonging to 18 different serogroups showed at least presence of one virulence gene studied. Twenty-eight strains (16.47%) (belonging to 13 different serogroups) from local goats carried stx(1) gene alone or in combination with stx(2) gene, while as only one strain (2%) from migratory goats possessed stx(2) gene alone. Interestingly in the present study none of the STEC strains carried eaeA gene. Similarly, none of the strains from local goats possessed eaeA and none of the migratory goats possessed stx(1) gene. Eight strains (16%) (belonging to four different serogroups) from migratory goats carried eaeA gene. Twenty-five (14.7%) and seven (14%) strains from local and migratory goats harboured hlyA gene respectively. CONCLUSIONS: Healthy goats of Jammu and Kashmir state serve as a reservoir of STEC and EPEC. Further studies in this direction are needed to work out whether or not they are transmitted to humans in this part of world. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first report of isolation of STEC and EPEC strains from healthy goats in Jammu and Kashmir State of India, which could be a source of infection to humans.     

Penicillin-binding proteins of pathogenic Escherichia coli strains

The penicillin-binding proteins of 11 pathogenic Escherichia coli strains, including enteropathogenic, enterotoxigenic, enteroinvasive, enteroaggregative, and enterohemorrhagic E. coli, were detected in gels following the labeling of isolated cell envelopes with [³H]benzylpenicillin. The electrophoretic profiles, sensitivities to and morphological changes induced by β-lactam antibiotics showed that the penicillin-binding proteins of most pathogenic E. coli possess structural and physiological functions similar to those of E. coli K12.     

Proteomic analysis of uropathogenic Escherichia coli

Urinary tract infections (UTIs) are among the most common of bacterial infections in humans. Although a number of Gram-negative bacteria can cause UTIs, most cases are due to infection by uropathogenic E. coli (UPEC). Genomic studies have shown that UPEC encode a number of specialized activities that allow the bacteria to initiate and maintain infections in the environment of the urinary tract. Proteomic analyses have complemented the genomic data and have documented differential patterns of protein synthesis for bacteria growing ex vivo in human urine or recovered directly from the urinary tracts of infected mice. These studies provide valuable insights into the molecular basis of UPEC pathogenesis and have aided the identification of putative vaccine targets. Despite the substantial progress that has been achieved, many future challenges remain in the application of proteomics to provide a comprehensive view of bacterial pathogenesis in both acute and chronic UTIs.     

一种用质粒DNA转化大肠杆菌感受态细胞的实用操作技巧

目的是建立一种简化、实用的用质粒DNA转化大肠杆菌的操作方法.采用氯化钙法制备大肠杆菌感受态细胞.以质粒pUC18,pCSN44,pAN52-1Not,pETts,pANth和植物双元表达栽体pCAMBIA1301分别转化用于质粒扩增与保存的常用大肠杆菌菌株Top10和DH5α以及用于原核表达的常用大肠杆菌菌株BL21(DE3)和TB1.质粒与感受态细胞的混合液置冰上作用一定时间后,直接涂布含有筛选抗生素的LB平板,于37℃培养12～16h.结果表明,用不同大小的质粒DNA转化不同的大肠杆菌菌株,都可以获得满足实验要求,转化效率可高达103～4阳性克隆/μg.该方法较标准的转化流程更加简便、省时、实用.     

A comparison of enteropathogenic and enterohaemorrhagic Escherichia coli pathogenesis

This review covers enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) infections, focusing on differences in their virulence factors and regulation. While Shiga-toxin expression from integrated bacteriophages sets EHEC apart from EPEC, EHEC infections often originate from asymptomatic carriage in ruminants whereas human EPEC are considered to be overt pathogens and more host-restricted. In part, these differences reflect variation in adhesin repertoire, type III-secreted effectors and the way in which these factors are regulated.     

Virulence-related DNA sequences and adherence patterns in strains of enteropathogenic Escherichia coli

The presence of the genes for Escherichia coli adherence factor (EAF), attaching and effacing lesion (eae) and bundle-forming pili (bfp) in 72 strains identified as enteropathogenic E. coli (EPEC) by slide agglutination was evaluated using hybridization and PCR. The adherence property of these strains was assayed using 3h HeLa cells adherence assay. The results obtained indicated that virulence-associated genes were present in 65% of the strains but only ten (13.9%) isolates were positive for all the three markers (typical EPEC), 37 (51.4%) isolates carried either one or two of these determinants (atypical EPEC) and the remaining 25 (34.7%) were negative for all these genes. In vitro adherence assay showed that 44 (61.1%) strains adhered to HeLa cells with a defined pattern, 13 (18.1%) isolates adhered loosely with no definite pattern and the remaining 15 (20.8%) were non-adherent. Analysis of the results showed a statistically significant association between the presence of the virulence-related genes with adherence of the strains with a defined pattern (P相似文献   

Cloning of the ethidium efflux gene from Escherichia coli

The gene specifying the ethidium efflux system of Escherichia coli has been cloned on a 3.2 kbp HindIII fragment and located on a 1.2 kbp fragment within this. Cross-resistance studies indicate that the system has a broad specificity for monovalent cations and the gene shows no hybridisation with similar genes found in Staphylococci.     

大肠埃希菌蛋白指纹图谱的建立

目的建立大肠埃希菌（Escherichia coli,E．coli）蛋白指纹图谱,为Ecoli感染快速诊断奠定基础。方法收集临床分离E.coli88株,提取细菌DNA,PCR检测Ecoli 16S rRNA。蛋白提取液提取细菌蛋白,干化学法测蛋白浓度,应用表面增强激光解析电离飞行时间质谱技术（SELDI-TOF-MS）检测Ecoli蛋白,采用Ciphergen Pro-teinchip软件自动采集数据。重复测定20次Ecoli混合标本,评价SELDI检测Ecoli蛋白分子量的重复性。结果E．coil标准菌株ATCC 25922和临床分离株均可检出16S rRNA。AU芯片能捕获近30个E．coli蛋白峰,其中19个蛋白峰构成E．coli特征性蛋白指纹图谱,各蛋白峰在临床分离E．coli间分子量变异系数≤0．2％。SELDI重复检测20次E．coli混合标本显示同一蛋白峰的分子量变异系数≤0．05％。结论E．coli在分子量3～20kD范围内具有特征性蛋白指纹图谱,为快速诊断E.coli感染提供了新思路。     

大肠杆菌血凝反应的研究

本文报告不同来源大肠杆菌对不同红血球的血凝反应。使用Evans法,磷酸盐缓冲液琼脂在4℃环境中操作可获满意的结果。人粪源大肠杆菌与豚鼠红血球的MRHA为27.3％,而尿源菌株仅2.7％。人和猪粪源大肠杆菌对A型红血球的MRHA为0～4％,而人尿源株为41.3％。尿源菌株对P_2~K血球MRHA为41.3％,对血球为12％。尿源菌株对A型与P_2~K型红血球的MRHA相符率达97％,扫描电镜显示具有菌毛的细菌粘附于A型红血球表面,A型红血球可取代罕有的P_2~K血球作血凝试验以诊断尿道致病性大肠杆菌。     

原核系统可溶性表达策略

获得大量目的蛋白的最简单最经济的方法是利用原核表达系统表达外源基因.但由于原核系统的自身特点,使所表达的蛋白常常形成无活性的包涵体.多年来世界各国的研究为解决这一问题尝试了多种方法.本简单介绍原核表达系统的特点及提高蛋白可溶性表达的常用方法.     

致病性大肠埃希菌血清型分布及对抗生素的敏感性分析

目的了解临床病例中致病性大肠埃希菌的主要血清型和对抗生素的敏感性。方法致病性大肠埃希菌的鉴定使用血清学的方法,药敏试验采用纸片扩散法,WHONET 5.0软件分析药敏结果。结果致病性大肠埃希菌的检出率为5.93%,共分离到7种血清型。在分离到的菌株中,ESBLs的检出率达45%。结论致病性大肠埃希菌是引起小儿腹泻的一种重要致病菌,应开展对致病性大肠埃希菌的检测,根据药敏结果选用合适药物。     

Characterization of enteroaggregative Escherichia coli isolates

Forty enteraggregative Escherichia coli (EAggEC) previously characterized by their ability to adhere to HEp-2 cells or/and their hybridization with the 1-kb EAggEC DNA probe were investigated for the presence of adherence factors and heat-stable enterotoxin (EAST1)-encoding genes. Only 45% of the isolates harbored the EAST1-encoding genes as detected by polymerase chain reaction. None of them hybridized with an AAF/II-encoding gene specific DNA probe and 35% (14/40) were positive in a PCR assay using primers specific for aggC, an accessory gene of the AAF/I-encoding operon. Cloning and sequence analysis of the aggA variant from one isolate, EAggEC 457, revealed 68.9% identity between its deduced amino acid sequence and those of the aggA product from the AAF/I-producing reference strain, E. coli 17.2. No major protein subunit was detected at the surface of EAggEC 457 compared to the bacterial surface extract of E. coli 17.2.