Evidence for a net-like organization of lipopolysaccharide particles in the Escherichia coli outer membrane

Cell wall LPS of Escherichia coli are organized as particles which are visible in the electron microscope, after treatment of the wall with alkali. We now describe alkali treated walls of three E. coli strains with differences in susceptibility to the T4 phage infection. Strain CR63, a usual host for the T4 phage, shows the LPS particles on the murein layer. These particles are absent in alkali treated cell walls of the strain W. Walls of this strain are broken during T4 infection and phages can be seen bearing pieces of membrane attached to their long as well as their short tail fibers. Strain AS19 which is hypersensitive to the lysis from without caused by T4 shows murein layers with no LPS particles on their surface, and networks of LPS particles with bacterial shape. This suggested that LPS are organized in a network of particles which may serve as the skeleton of the cell wall.     

Human antibody responses to R3-core epitopes on the lipopolysaccharide of Escherichia coli O157

AIMS: To establish the incidence of serum antibodies binding to the R3-core lipopolysaccharide (LPS) of verocytotoxin-producing Escherichia coli (VTEC) O157, in patients with serum antibodies to E. coli O157 LPS, and to characterize the class(es) of antibodies binding to epitopes on the R3-core. METHODS AND RESULTS: SDS-PAGE profiles of LPS prepared from VTEC O157 were used in combination with immunoblotting to detect and characterize serum antibodies binding to the R3-core LPS of VTEC O157. Of 417 sera, referred to the Laboratory of Enteric Pathogens (LEP) for routine O157 serology and found to have serum antibodies to long-chain VTEC O157 LPS, 31 had antibodies binding to the R3-core of VTEC O157 LPS. The majority of the 31 sera contained IgA-class antibodies to both long-chain and R3-core LPS epitopes. Patients who did not develop haemolytic uraemic syndrome (HUS) produced antibodies of the IgM class to R3-core and IgG-class antibodies to long-chain LPS more frequently than patients with HUS. CONCLUSIONS: Only 7.4% of sera received by the LEP, and shown to have antibodies to VTEC O157 LPS, contained antibodies binding to the R3-core of VTEC LPS. Most sera contained IgA-class antibodies to both long-chain and R3-core LPS epitopes. SIGNIFICANCE AND IMPACT OF THE STUDY: Patients infected with VTEC O157 produced antibodies binding to the R3-core epitopes of VTEC O157 LPS only rarely, and these antibodies are unlikely to interfere with the serodiagnosis of infections caused by these organisms.     

EDTA induced autolysis in Escherichia coli and isolation of resistant mutants

Abstract Autolysis of Escherichia coli induced by a shock treatment with 10⁻³M EDTA, pH 6.5 was investigated. Mutants presenting reduced rates of EDTA-induced autolysis were isolated. A remarkable feature of these mutants was their tolerance to penicillin G, cephaloridine and moenomycin. Furthermore, a reduced level of peptidoglycan endopeptidase or N -acetylmuramidase activity was observed. Penicillin-binding protein patterns were unaltered.     

Permeabilizing effects of sub-inhibitory concentrations of microcystin on the growth of Escherichia coli

Microcystin, a cyanotoxin produced by Microcystis aeruginosa, lacks potent antibacterial activity. When tested in combination, in vitro, inhibitory values for a range of hydrophobic antibiotics were significantly reduced in the presence of at least 1/20 x the minimum inhibitory concentration of microcystin. The degree of inhibition was equivalent to that of a well-characterised permeabilizing agent, polymyxin B nonapeptide. The permeabilizing ability of sub-inhibitory concentrations of microcystin to affect the envelope of Escherichia coli was demonstrated by a rapid and sustained reduction in absorbance values of lysozyme-treated cells and by enhanced uptake of crystal violet in microcystin-treated cultures. Direct effects of appropriate concentrations of microcystin on the integrity of bacterial outer and inner membranes were measured by release of specific enzyme markers. Although the exact mechanism for permeabilizing E. coli with microcystin has not been elucidated, the effects were consistent with permeability changes to the enterobacterial outer membrane caused by polymyxin B nonapeptide.     

禽致病性大肠杆菌脂多糖核心型分布与毒力基因的相关性分析

【目的】大肠杆菌脂多糖(LPS)核心型根据其化学结构的不同分为5种,即R1、R2、R3、R4和K12。通过对禽致病性大肠杆菌(Avian pathogenic Escherichia coli,APEC)安徽、江苏、上海和河南等省市分离株的脂多糖核心型分布情况的研究,分析其与大肠杆菌主要毒力基因之间的潜在联系,以期为APEC的研究和防治提供参考。【方法】对分离到的76株APEC,利用PCR方法开展对LPS核心型分型鉴定和毒力基因检测;分析LPS核心型的分布和毒力基因、致病性之间的相关性。【结果】在76株APEC分离株中,68.4% (52株)为R1核心型,15.8% (12株)为R3型,11.8% (9株)为R4型,3.9% (3株)为R2型,未检测到K12核心型。毒力基因鉴定结果中yijp、mat、fimC、ibeB和ompA的检验阳性率均达到90%以上,可作为APEC的保守基因。其中LPS核心型R1与neuC、cva/cvi、irp2均具有显著正相关性(P<0.05),R3与iroN、irp2均具有显著负相关性(P<0.05),R4核心型与aatA显著正相关(P<0.05)。【结论】APEC的LPS核心型主要为R1。LPS核心型对部分毒力基因分布具有显著影响。     

Inhibitors of lipopolysaccharide biosynthesis impair the virulence potential of Escherichia coli

Inhibition of 3-deoxy-manno-octulosonate cytidylytransferase (CMP-KDO transferase; EC 2.7.7.38) by 8-amino-2,6-anhydro-3,8-dideoxy-D-glycero-D-talo-octonic acid (NH2dKDO) halts the growth of Gram-negative bacteria by depriving the cells of the 3-deoxy-D-manno-2-octulosonate required for the biosynthesis of the core region of the lipopolysaccharide components of the outer membrane. Low levels of this inhibitor increase the vulnerability of Escherichia coli to hydrophobic antibiotics, detergents, the complement-mediated antibacterial activity of serum, phagocytosis, and enhance the rate at which bacteria are cleared from the mouse bloodstream.     

大肠杆菌细胞外膜渗透性与脂多糖结构的关系

细胞外膜是大肠杆菌的半透膜屏障, 其主要成分是脂多糖。选取并构造共9种具有不同脂多糖结构的大肠杆菌, 用于考察脂多糖结构对细胞外膜渗透性的影响。从9种菌株中提取出脂多糖和类脂A, 并且用薄层层析色谱和离子源质谱来鉴定其结构。用N-苯基-1-萘胺作为荧光探针来测定细胞外膜渗透性大小。野生型大肠杆菌表现出最小的渗透性, 因敲除或表达某些基因而导致脂多糖结构改变的突变株均表现出较高的渗透性。脂多糖上的磷酸基团、脂肪酸链和多糖链的改变都影响了大肠杆菌的渗透性, 其中多糖链长度的改变对渗透性影响最大, 其次是脂肪酸链的数目变化。实验结果表明渗透性和脂多糖的结构具有较强的相关性。     

Effects of ethanol on the growth and elongation of Escherichia coli under high pressures up to 40 MPa

The effects of ethanol on growth, viability and cell length were studied in Escherichia coli cultured under pressures up to 40 MPa (400 bar). A pressure of 10 MPa reversed the effect of ethanol in retarding cell growth. Cells cultured in the absence of ethanol became about seven times longer at 40 MPa than at atmospheric pressure, and some cells showed incomplete cell division. Ethanol also increased cell length but these effects were not seen at pressures of 20 MPa or more.     

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.     

Plasmid Effects on Escherichia coli Metabolism

ABSTRACT:?

The idea that plasmids replicate within hosts at the expense of cell metabolic energy and preformed cellular blocks depicts plasmids as a kind of molecular parasites that, even when they may eventually provide plasmid-carrying strains with growth advantages over plasmid-free strains, doom hosts to bear an unavoidable metabolic burden. Due to the consistency with experimental data, this idea was rapidly adopted and used as a basis of different hypotheses to explain plasmid-host interactions. In this article we critically discuss current ideas about plasmid effects on host metabolism, and present evidence suggesting that the complex interaction between plasmids and hosts is related to the alteration of the cellular regulatory status.     

基因atpA、purHD和ndh的过量表达对大肠杆菌DH5a生长的影响

【目的】理解大肠杆菌DH5a及其耐乙酸突变株DA19在基本培养基中生长和乙酸积累差异的机理。【方法】根据前期针对两个菌株蛋白质组和培养中物料及能量平衡的分析,通过PCR分别扩增大肠杆菌DH5a及DA19的atpIBEFHAGDC基因、purHD基因和与NADH氧化代谢相关ndh基因及其各自的调节区,以及NADH脱氢酶Ⅰ基因（nuoA-N）的调节区。将atpA、purHD和ndh基因分别克隆到载体pTrc99a中,转化DH5a,研究基因过量表达对生长的影响。【结果】测序结果表明两个菌株的这些DNA序列与公布的大肠杆菌K-12的相应序列完全一致,在DH5a菌株中分别过量表达atpA、purHD或ndh基因可以在一定程度上改善菌体的生长。【结论】过量表达purHD或ndh基因比过量表达atpA基因的效果更好,但与DA19的生长相比仍然存在较大差距,反映了代谢全局调节的重要性。     

Effect of growth rate on the penicillin-binding proteins of Escherichia coli

Abstract In an Escherichia coli strain, the levels of penicillin-binding proteins (PBPs) 1A plus 1B, both peptidoglycan transglycosylase/transpeptidases, were found to be relatively independent of the imposed growth ratw in chemostat cultures under different nutrient limitation conditions. A considerable increase in levels of PBP 6 was observed as the growth rate was reduced, whilst, in contrast, a decrease was observed in levels of the other PBPs.     

His-tag对胆固醇氧化酶基因在大肠杆菌中表达的影响

为考察组氨酸标签(His-tag)对Brevibacterium sp.DGCDC-82中胆固醇氧化酶基因(ChoAb)在大肠杆菌中表达的影响,将PCR扩增后得到的结构基因与pET28a(+)连接,构建重组质粒pETChoAb(不带His-tag),pETChoAbn(His-tag位于N端)和pETChoAbc(His-tag位于C端)并在大肠杆菌中进行表达.对重组酶进行酶活检测,结果表明His-tag位于ChoAb的C端和N端,COD单位体积酶活由未带标签时的1.72 U/mL分别提高到4.03 U/mL和11.36 U/mL.利用软件Quantity One对SDS-PAGE电泳条带进行灰度分析,结果显示与不带His-tag的COD相比,His-tag位于ChoAb的C端和N端,COD表达量由8.8％增加到16.4％与72.3％.同时菌体浓度分别提高了1.2倍和3.2倍.作为纯化标签,该研究结果对His-tag用于诊断用酶COD的分离纯化可以提供一定的理论指导.     

Inhibition of growth of Shiga toxin-producing Escherichia coli by nonpathogenic Escherichia coli

During routine quality control testing of diagnostic methods for Shiga toxin-producing Escherichia coli (STEC) using stool samples spiked with STEC, it was observed that the Shiga toxin could not be detected in 32 out of 82 samples tested. Strains of E. coli isolated from such stool samples were shown to be responsible for this inhibition. One particular isolate, named E. coli 1307, was intensively studied because of its highly effective inhibitory effect; this strain significantly reduced growth and Shiga toxin levels in coculture of several STEC strains regardless of serovar or Shiga toxin type. The probiotic E. coli Nissle 1917 inhibited growth and reduced Shiga toxin levels in STEC cultures to an extent similar to E. coli 1307, but commensal E. coli strains and several other known probiotic bacteria (enterococci, Bacillus sp., Lactobacillus acidophilus ) showed no, or only small, inhibitory effects. Escherichia coli 1307 lacks obvious fitness factors, such as aerobactin, yersiniabactin, microcins and a polysaccharide capsule, that are considered to promote the growth of pathogenic bacteria. We therefore propose strain E. coli 1307 as a candidate probiotic for use in the prevention and treatment of infections caused by STEC.     

Characteristics of sheep-rumen isolates of Pseudomonas aeruginosa inhibitory to the growth of Escherichia coli O157

Screening facultative sheep-rumen bacteria which inhibit growth of Escherichia coli produced 11 strains of Pseudomonas aeruginosa. The isolates showed three different pulsed-field gel electrophoresis patterns and strains from different sheep produced pyocins that varied in strain specificity. Representative strains were resistant to ampicillin, methicillin, erythromycin, fusidic acid and augmentin, but not to tetracycline or nalidixic acid. Tested strains attached in large numbers to cultured rumen epithelial cells, potentially providing a means of survival in this ecosystem.     

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.     

抗菌肽OH-CATH对大肠杆菌Escherichia coli的作用(英文）

OH-CATH是眼镜王蛇中新发现的cathelicidin家族抗菌肽.它在1%NaCI存在的条件下对多种细菌都有较强的抗菌活性,同时,在高浓度下对人红细胞无溶血活性.OH-CATH足开发新型抗菌药物的优良模板.蜊明OH-CATH的作用机理及其对微生物的选择性,对研发以OH-CATH为先导结构的药物研发有十分重要的意义.本文利用扫描电镜以及透射电镜对OH-CATH与革兰氏阴性菌一大肠杆菌ATCC 25922相互作用的效应研究.结果揭示:OH-CATH对大肠杆菌的作用涉及到3个步骤.首先,OH-CATH借助其带正电的氨基酸残基附着到细菌带负电荷的细胞壁:然后,附着的OH-CATH在达剑一定浓度后发生聚集,以孔道彤成的方式破坏细菌的膜结构;最终,由十细菌膜的损坏,膜的渗透性被破坏,胞内内含物释放造成细菌死亡.     

ppc基因敲除对大肠杆菌L-色氨酸发酵的影响

目的：减少大肠杆菌L-色氧酸前体物质磷酸烯醇式丙酮酸向草酰乙酸的代谢流,提高其L-色氨酸的产量。方法：以大肠杆菌TRTH0709为出发菌株,利用Red重组敲除技术敲除磷酸烯醇式丙酮酸羧化酶（Ppc）编码基因PPc,并经测序和酶活性检测确证;对出发菌株和基因敲除菌株进行L-色氖酸发酵,对比分析发酵结果。结果：测序和酶活性检测结果表明ppc基因被成功敲除。发酵结果表明,与出发菌株相比,基因敲除菌株TRTH0709△ppc生长速度减慢,最终生物量减少32％,L-色氯酸产量降低27％,但糖酸转化率提高6％;向发酵培养基中添加1％琥珀酸后,TRTH0709△ppc的生长速率和产酸量有所提高,但仍与出发菌株有一定差距。结论：虽然ppc基因敲除对菌体生长和产酸量影响较大,但能有效提高其糖酸转化率;选育Ppc弱化的突变株以达到减弱代谢流且不影响菌体生长,以及增加,L-色氨酸积累的目的,将是本研究今后的主要方向。