Involvement of oxidative stress in hydroquinone-induced cytotoxicity in catalase-deficient Escherichia coli mutants

Hydroquinone is a benzene-derived metabolite. To clarify whether the reactive oxygen species (ROS) are involved in hydroquinone-induced cytotoxicity, we constructed transformants of Escherichia coli (E. coli) strains that express mammalian catalase gene derived from catalase mutant mice (Cs^b, Cs^c) and the wild-type (Cs^a) using a catalase-deficient E. coli UM255 as a recipient. Specific catalase activities of these tester strains were in order of Cs^a > Cs^c > Cs^b > UM255, and their susceptibility to hydrogen peroxide (H₂O₂) showed UM255 > Cs^b > Cs^c > Cs^a. We found that hydroquinone exposure reduced the survival of catalase-deficient E. coli mutants in a dose-dependent manner significantly, especially in the strains with lower catalase activities. Hydroquinone toxicity was also confirmed using zone of inhibition test, in which UM255 was the most susceptible, showing the largest zone of growth inhibition, followed by Cs^b, Cs^c and Cs^a. Furthermore, we found that hydroquinone-induced cell damage was inhibited by the pretreatment of catalase, ascorbic acid, dimethyl sulfoxide (DMSO), and ethylenediaminetetraacetic acid (EDTA), and augmented by superoxide dismutase (both CuZnSOD and MnSOD). The present results suggest that H₂O₂ is probably involved in hydroquinone-induced cytotoxicity in catalase-deficient E. coli mutants and catalase plays an important role in protection of the cells against hydroquinone toxicity.     

Isolation and preliminary characterization of β-lactamase excretory mutants of Escherichia coli K-12

Abstract Escherichia coli exc mutants able to release the plasmid pBR322-encoded β-lactamase (EC 3.5.2.6) into the extracellular medium have been isolated using a new in situ plate assay.
A preliminary characterization of the exc mutants was carried out: the presence of exc mutations was associated with a specific or pleiotropic pattern of excretion of periplasmic enzymes, an increased sensitivity to different growth inhibitors (EDTA, chloramphenicol, cholic acid) and a poor growth on various carbon sources.
After quantitative analysis, three groups of exc mutants were identified on the basis of their temperature-dependent or -independent pattern of growth and β-lactamase synthesis and excretion.     

Porins and lipopolysaccharide of Escherichia coli ATCC 25922 and isogenic rough mutants

Abstract The lipopolysaccharide and porin profile of Escherichia coli ATCC 25922, a smooth strain commonly used in antibiotic susceptibility testing, and five isogenic rough mutants was examined. The lipopolysaccharide of the parent strain had the characteristic ladder pattern on polyacrylamide gels, while that of the mutants appeared similar to chemotypes Ra and Rc of Salmonella typhimurium with some changes in chemical composition. Of the porins, OmpC appeared markedly reduced in the parent strain while OmpF appeared markedly reduced in the mutants. In addition, a new outer-membrane protein of size intermediate to that of OmpC and OmpF was detected in all mutants. Neither parent nor mutants were susceptible to the LPS core-specific P1 phage or the porin-specific PA2 and K20 phages.     

Effects of sub-minimal inhibitory concentrations of EDTA on growth of Escherichia coli and the release of lipopolysaccharide

Abstract Release of lipopolysaccharide from E. coli was studied in the presence of sub-minimal inhibitory concentrations of ethylenediaminetetraacetic acid (EDTA). In untreated cells no release was detected with 50 mM Mg²⁺ in the medium, but a steady release of over 50% of the synthesized lipopolysaccharide was observed with 0.1 mM Mg²⁺. EDTA at MIC/8 led to a 2- to 3-fold higher release, presumably by an adjustment of the concentration of unchelated Mg²⁺ to a value still sustaining normal growth but giving rise to a highly unstable outer membrane. No structural difference was observed between cell-bound and released lipopolysaccharide.     

Relaxed mutants of Escherichia coli RNA polymerase

When Escherichia coli cells are treated with either polymixin or gramicidin at concentrations that block protein and RNA synthesis, they accumulate a significant amount of guanosine tetraphosphate ppGpp. Such accumulation occurs in stringent (relA+) as well as in relaxed (relA) strains and no guanosine pentaphosphate pppGpp is then detected within the cells. These observations suggest that polypeptide antibiotics elicit ppGpp formation through a mechanism different from the stringent control system triggered by amino acid starvation of bacteria. Experiments based on tetracycline action indicate, moreover, that the accumulation of ppGpp under polymixin or gramicidin treatment is connected with a strong restriction of the degradation rate of this nucleotide.     

Uptake and degradation of EDTA by Escherichia coli

It was found that Escherichia coli exhibited a growth by utilization of Fe(III)EDTA as a sole nitrogen source. No significant growth was detected when Fe(III)EDTA was replaced by EDTA complexes with other metal ions such as Ca²⁺, Co²⁺, Cu²⁺, Mg²⁺, Mn²⁺, and Zn²⁺. When EDTA uptake was measured in the presence of various ions, it was remarkable only when Fe³⁺ was present. The cell extract of E. coli exhibited a significant degradation of EDTA only in the presence of Fe³⁺. It is likely that the capability of E. coli for the growth by utilization of Fe(III)EDTA results from the Fe³⁺-dependent uptake and degradation of EDTA.     

Response dynamics of 26-, 34-, 39-, 54-, and 80-kDa proteases in induced cultures of recombinant Escherichia coli

Several researchers have demonstrated that the presence of a heterologous protein in recombinant Escherichia coli elicits a response similar to the heat-shock response, which includes enhanced protease expression. The present work detects, quantifies, and characterizes intracellular protease activity in E. coli that are "shocked" by the induction of a recombinant protein, CAT, which is an endogenous protein in some E. coli strains. A novel, sodium dodecyl sulfate gelatin poly-acrylamide gel electrophoresis (SDS-GPAGE) method is used to detect, quantify, and characterize the presence of these proteases. A hypothesis is proposed which links the amplified protease activity to a temporary depletion of specific amino acid pools, and a stringent-like stress response. (c) 1993 John Wiley & Sons, Inc.     

大肠杆菌磷酸甘油酸脱氢酶突变体的构建及抗反馈抑制效应

磷酸甘油酸脱氢酶(D-3-phosphoglycerate dehydrogenase,PGDH,EC 1.1.1.95)为L-丝氨酸合成途径的关键酶,其编码基因为ser A,其活性受到合成产物L-丝氨酸的反馈抑制调控。为解除丝氨酸的反馈抑制,采用定点突变技术把编码PGDH酶344位组氨酸或346位天冬氨酸或364位天冬氨酸的密码子定点突变为丙氨酸密码子。改造后的ser AFbr被连到表达载体pT7-7上,并转入大肠杆菌Escherichia coli BL21(DE3)中进行表达,破壁回收粗酶液,通过DEAE阴离子柱纯化PGDH突变体,并对其酶活性和IC_(50)值进行了测定。结果,野生型PGDH酶IC_(50)值为7μmol/L,而PGDH双突变体N346A/H344A催化活性与野生型相近,在丝氨酸浓度为160 mmol/L时,其酶活仍保持未添加丝氨酸时酶活的96%,基本解除反馈抑制。     

大肠杆菌murB和birA基因的转录通读现象

在对应用限制性显示 PCR技术构建的大肠杆菌 poly(A)化mRNA的cDNA文库鉴定中 ,发现了一克隆的基因片段同时跨越了murB和birA基因的部分序列 ,提示这 2个基因存在转录通读现象 ,设计引物 ,利用逆转录后的产物为模板 ,进行了验证。结果表明murB和birA基因存在转录通读现象。     

The expression of an R3 lipopolysaccharide-core by pathotypes of Escherichia coli

AIMS: To investigate the incidence of an R3 lipopolysaccharide (LPS)-core amplicon in a range of pathotypes of Escherichia coli, including Verocytotoxin-producing E. coli (VTEC), enteroaggregative E. coli (EAggEC) and enteropathogenic E. coli (EPEC). METHODS AND RESULTS: A total of 100 strains of E. coli belonging to a range of pathotypes, including 41 strains of VTEC, were screened for the genes encoding the R3 LPS-core using PCR. Fifty-four per cent produced an amplicon with the R3 primer set. Of the 41 VTEC, 66% had an R3 LPS-core with a PCR product being observed with all strains belonging to serotypes O26:H11, O111ac:H- and O145:H25. However, 46% of enteroaggregative E. coli and 50% of enteropathogenic E. coli were also shown to have an R3 LPS-core structure. CONCLUSIONS: Strains with an R3 LPS-core are widely distributed within the species E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: Strains of E. coli with an R3 LPS-core structure appear not to be associated with a specific pathotype.     

Small GTP-binding proteins of the Rho- and Ras-subfamilies are not involved in the actin rearrangements induced by attaching and effacing Escherichia coli

Attaching and effacing Escherichia coli (AEEC) are extracellular pathogens that induce the formation of actin-rich structures at their sites of attachment to eukaryotic host cells. We analysed whether small GTP-binding proteins of the Rho- and Ras-subfamilies, which control the cellular actin system, are essential for these bacterial-induced microfilament reorganizations. For this purpose we specifically inactivated them using the Clostridium difficile toxins TcdB-10463 and TcdB-1470. Such treatment led to a dramatic breakdown of the normal actin cytoskeleton, but did not abrogate the bacterial-induced actin rearrangements. Our data therefore indicate that the microfilament reorganizations induced by AEEC are independent of those small GTP-binding proteins that under normal conditions control the dynamics and maintenance of the actin cytoskeleton.     

大肠杆菌梭曼水解酶的纯化和性质

大肠杆菌梭曼水解酶的纯化和性质邵煌,刘昌玲,肖美珍,孙曼霁（北京军事医学科学院毒物药物研究所,北京１００８５０）梭曼属Ｇ类神经性有机磷毒剂．自然界发现多种细菌中均存在梭曼水解酶（Ｓｏｍａｎａｓｅ）活性［１－３］．研究细菌梭曼水解酶,寻求生物解毒的方法...     

猪源大肠杆菌(ETEC、STEC、AEEC)毒力基因及其与O抗原型的关系

[目的]揭示从我国部分地区仔猪腹泻或水肿病病猪体内分离到的300个大肠杆菌分离株所属病原型(pathotype)、毒力基因及其与O血清型的关系.[方法]O血清型采用常规的凝集试验进行测定,毒力基因采用PCR方法检测.[结果]通过对这300个分离株的O血清型及其毒素、紧密素和黏附素基因进行鉴定,结果显示除50株未定型、17株自凝外,测定出233个分离株的血清型,这些分离株覆盖了45个血清型,其中以0149、0107、0139、093和091为主,共133株,占定型菌株的57.1%;拥有est Ⅰ、estⅡ、elt、stx2e和eae A基因的菌株分别为102(34.0%)、190(63.3%)、81(27.0%)、57(19.0%)和54(18.0%)株;分离株中有51株K88基因阳性(其中菌毛表达率为100%),75株F18基因阳性(其中菌毛表达率为50.7%),在K88菌株中,0149血清型与est Ⅰ或estⅡ elt密切相关,在F18菌株中,0107血清型与est Ⅰ或estⅡ、0139血清型与stx2e紧密相关.依其毒力特征可将这些分离株分为以下6种类型:ETEC、STEC、AEEC、ETEC/STEC、AEEC/ETEC和AEEC/ETEC/STEC,分别拥有190、24、36、32、17和1个菌株,占分离株的63.3%、8.0%、12.0%、10.7%、5.7%和0.3%.通过分析这些分离株的O血清型、毒素类型和黏附素型之间的相关性:猪源ETEC以0149、0107、093和098等血清型为主,0149:K88菌株主要与estⅡ或estⅡ elt肠毒素相关,0107:F18菌株主要与estⅡ相关,093和098血清型菌株主要与estⅡ肠毒素相关;STEC菌株以0139:F18血清型为主,拥有stx2e;AEEC菌株拥有紧密素,无明显优势血清型;ETEC/STEC菌株以0107:F18和0116:F18血清型为主,主要与est Ⅰ stx2e或estⅡ stx2e密切相关,ETEC/AEEC菌株以091和0107血清型为主,全部拥有肠毒素est Ⅰ和紧密素基因.[结论]我国至少存在6种病原型的猪肠道致病性大肠杆菌,其中ETEC为我国部分地区猪大肠杆菌病的主要病原,同时其病原型日益复杂.     

Production and characterization of Escherichia coli enterohemolysin and its effects on the structure of erythrocyte membranes

Hemolysins are cell-damaging protein toxins produced by pathogenic bacteria, which are usually released into the extracellular medium. Escherichia coli enterohemolysin is an intracellular toxin produced during the log phase of growth, with a maximal intracellular accumulation in the late log phase. In the present study, we have employed electron microscopy and SDS-PAGE to assess the effects of enterohemolysin on erythocyte membranes from different species. The erythrocyte cell damage began immediately after exposure to enterohemolysin with chemically detectable changes in cell membrane permeability, and the formation of surface lesions which increased rapidly in size. This process resulted in complete cell destruction. Ring-shaped structures with a diameter of 10nm were observed by electron microscopy after treatment of horse erythrocyte membranes with enterohemolysin. The ring structures were found clustered and irregularly distributed on the surface of the membranes. Following incubation of the toxin with horse erythrocyte ghosts and detergent-solubilization, the enterohemolysin was isolated from the cytoplasm in its membrane-bound form by sucrose density gradient. SDS-PAGE and silver staining of deoxycholate-solubilized target membranes revealed heterogeneous forms of the toxin. By using SDS-PAGE and gel filtration, the molecular weight of the toxin was estimated to be 35 kDa. With respect to species specificity, horse erythrocytes showed the highest sensitivity to the enterohemolysin, followed by human and guinea pig erythrocytes. The hemolytic sensitivity correlated with the toxin binding capacity of erythrocyte membranes of different animal species. The degree of hemolysis was unaffected by temperature in the range of 4 degrees C-37 degrees C and was optimal at pH 9.0. In contrast to pore-forming cytolysins, the hemolytic activity of enterohemolysin was enhanced continuously in the presence of increasing concentrations of dextran 4 and dextran 8 within the range of 5 to 30 mM. Trypsin sensitivity of membrane-bound enterohemolysin indicates that the cell surface is the most likely target site for this toxin. Additionally, the fact that proteinase and phosphatase inhibitors failed to inhibit lysis suggests that enterohemolysin alters and disrupts cell membranes by a detergent-like mechanism.     

Uropathogenic Escherichia coli (UPEC) strains may carry virulence properties of diarrhoeagenic E. coli

To analyze whether Escherichia coli strains that cause urinary tract infections (UPEC) share virulence characteristics with the diarrheagenic E. coli (DEC) pathotypes and to recognize their genetic diversity, 225 UPEC strains were examined for the presence of various properties of DEC and UPEC (type of interaction with HeLa cells, serogroups and presence of 30 virulence genes). No correlation between adherence patterns and serogroups was observed. Forty-five serogroups were found, but 64% of the strains belonged to one of the 12 serogroups (O1, O2, O4, O6, O7, O14, O15, O18, O21, O25, O75, and O175) and carried UPEC virulence genes (pap, hly, aer, sfa, cnf). The DEC genes found were: aap, aatA, aggC, agg3C, aggR, astA, eae, ehly, iha, irp2, lpfA(O113), pet, pic, pilS, and shf. Sixteen strains presented aggregative adherence and/or the aatA sequence, which are characteristics of enteroaggregative E. coli (EAEC), one of the DEC pathotypes. In summary, certain UPEC strains may carry DEC virulence properties, mostly associated to the EAEC pathotype. This finding raises the possibility that at least some faecal EAEC strains might represent potential uropathogens. Alternatively, certain UPEC strains may have acquired EAEC properties, becoming a potential cause of diarrhoea.     

重组大肠杆菌的分批补料培养方法

在重组大肠杆菌的培养过程中,存在着菌体的高浓度与外源蛋白的高表达这一矛盾,使得重组菌的比生长速率通常远远低于宿主菌,限制了基因工程菌由实验室规模向工业化规模的转变。要实现重组大肠杆菌的高密度培养,最常用和最有效的方法就是分批补料流加培养。     

Incidence and survival of non-O157 verocytotoxigenic Escherichia coli in soil

Aims: This study estimated the incidence of non‐O157 verocytotoxigenic Escherichia coli (VTEC) in farm pasture soils and investigated the survival of non‐O157 VTEC in clay and sandy loam soils. Methods and Results: Twenty farms were tested over a 12‐month period by sample enrichment in tryptone soya broth plus vancomycin, followed by PCR screening for the presence of vt1 and vt2 genes. Of the 600 soil samples, 162 (27%), across all farms, were found to contain vt1 and/or vt2 genes. The enrichment cultures from the 162 PCR‐positive samples were plated onto Chromocult tryptone bile X‐glucuronide agar (TBX), presumptive VTEC colonies recovered, confirmed as VTEC by PCR and serotyped. Samples of the two predominant soil types in Ireland (clay and sandy) were homogenized, characterized in terms of pH, boron, cobalt, copper, potassium, magnesium, manganese, phosphorus, zinc and organic matter content, inoculated with washed suspensions of eight non‐O157:H7 soil isolates and six bovine faecal isolates and stored at 10°C for up to 201 days. Inoculum survival rates were determined at regular intervals by recovering and plating soil samples on TBX. All inoculated non‐O157 serotypes had highest D‐values in the sandy loam soil with D‐values ranging from 50·26 to 75·60 days. The corresponding range in clay loam soils was 31·60–48·25 days. Conclusions: This study shows that non‐O157 VTEC occur widely and frequently in pasture soils and can persist in such environments for several months, with considerable opportunity for recycling through farm environments, and cattle, with clear potential for subsequent transmission into the human food chain. Significance and Impact of the Study: This is the first such study of non‐O157 VTEC in farm soils and found that these VTEC are frequent and persistent contaminants in farm soils. In light of recent epidemiological data, non‐O157 VTEC should be seen as an emerging risk to be controlled within the food chain.