Mutations affecting pore formation by haemolysin from Escherichia coli

Summary By introduction of site-specific deletions, three regions in HlyA were identified, which appear to be involved in pore formation by Escherichia coli haemolysin. Deletion of amino acids 9–37 at the N-terminus led to a haemolysin which had an almost threefold higher specific activity than wild-type and formed pores in an artificial asolectin lipid bilayer with a much longer lifetime than those produced by wild-type haemolysin. The three hydrophobic regions (DI–DIII) located between amino acids 238–410 contributed to pore formation to different extents. Deletion of DI led to a mutant haemolysin which was only slightly active on erythrocyte membranes and increased conductivity of asolectin bilayers without forming defined pores. Deletions in the two other hydrophobic regions (DII and DIII) completely abolished the pore-forming activity of the mutant haemolysin. The only polar amino acid in DI, Asp, was shown to be essential for pore formation. Removal of this residue led to a haemolysin with a considerably reduced capacity to form pores, while replacement of Asp by Glu or Asn had little effect on pore formation. A deletion mutant which retained all three hydrophobic domains but had lost amino acids 498–830 was entirely inactive in pore formation, whereas a shorter deletion from amino acids 670–830 led to a mutant haemolysin which formed abnormal minipores. The conductivity of these pores was drastically reduced compared to pores introduced into an asolectin bilayer by wild-type haemolysin. Based on these data and structural predictions, a model for the pore-forming structure of E. coli haemolysin is proposed.     

Actinobacillus pleuropneumoniae haemolysin II is secreted from Escherichia coli by A. pleuropneumoniae pleurotoxin secretion gene products

Abstract Actinobacillus pleuropneumoniae serotype 2 secretes type II haemolysin and pleurotoxin activities. Here, the genes for type II haemolysin were cloned in Escherichia coli , but type II haemolysin antigen and haemolysin activity were only detected intracellularly and not exported to culture supernatant. It has been reported that the genes for type II haemolysin are not linked to functional secretion genes, while those for pleurotoxin are. In this report the means of secretion of type II haemolysis was examined by constructing a hybrid plasmid carrying the genes required for type II haemolysin expression, together with determinants which allow secretion of pleurotoxin and are linked to the pleurotoxin toxin genes. These genes facilitated the export of type II haemolysin from E. coli , and may perform this function in A. pleuropneumoniae .     

Antibacterial activity and pore-forming properties of ceratotoxins: a mechanism of action based on the barrel stave model

Ceratotoxins are α-helical cationic peptides isolated from the medfly Ceratitis capitata. These amphipathic peptides were found to display strong antibacterial activity and weak hemolytic activity. When reconstituted into planar lipid bilayers, ceratotoxins developed highly asymmetric I/V curves under voltage ramps and formed, in single-channel experiments, well-defined voltage-dependent ion channels according to the barrel stave model. The antibacterial activity and pore-forming properties of these molecules were well correlated. Similar experiments performed with synthesized truncated fragments showed that the C-terminal domain of ceratotoxins is strongly implicated in the formation of helical bundles in the membrane whereas the largely cationic N-terminal region is likely to anchor ceratotoxins on the lipid surface.     

Immunological cross-reactivity between outer membrane pore proteins of Campylobacter jejuni and Escherichia coli

Immunocrossreactivity between the major outer membrane protein (MOMP) of Campylobacter jejuni 85H and the OmpC porin of Escherichia coli K-12 was observed. These results indicate that a common antigenic domain is conserved in both MOMP and OmpC. This antigenic region is detected only after a 96 degrees C treatment suggesting that it is buried in the native conformation of the respective porins. In addition, differences were observed between the major outer membrane proteins from various C. jejuni strains. About 60% of the C. jejuni pathogenic strains tested contained a protein exhibiting a similar electrophoretic profile to the 85H porin.     

Comparison of the phenotypes of the lpxA and lpxD mutants of Escherichia coli

Abstract We compared the phenotype of two thermosensitive Escherichia coli mutants defective in lipid A biosynthesis i.e. SM101 ( lpxA ) and CDH23-213 ( lpxD ). More than 40% of the periplasmic 27-kDa marker enzyme β-lactamase was released from SM101 at 28°C. At this temperature, the mutant still grew with a generation time (67 min), not much longer than that of the parent control strain (57 min). CDH23-213 released β-lactamase only at higher temperatures. SM101 and CDH23-213 were both unable to grow in hypo-osmotic conditions. Derivatives of SM101 and CDH23-213 with mdoA ::Tn 10 had identical phenotypes (including thermosensitivity and defective outer membrane permeability barrier to hydrophobic probes) to those of SM101 and CDH23-213, indicating that the potential loss of membrane-derived oligosaccharides (MDO) did not explain these phenotypic properties. A method for the estimation of lipid A synthesis rate was developed.     

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.     

Electric field effects on membranes: gramicidin A as a test ground

Electric fields due to transmembrane potential differences or ionic gradients across the membrane are presumably crucial for many reactions across membranes or close to membranes like signal transduction, control of ion channels or the generation of neural impulses. Molecular dynamics simulations have been used to study the influence of external electric fields on a mixed gramicidin/phospholipid bilayer system. At high field strengths, formation of membrane electropores occurred both close and distal to the gramicidin. Gramicidin was found to stabilize the membrane adjacent to the protein but also at larger distances of up to 2-3 nm. As a result, membrane pore formation was found to be significantly suppressed for the mixed gramicidin/DMPC system. Moderate field strengths only weakly affected the structure and dynamics of the gramicidin. Spontaneous potassium passage events in external electric fields were observed for both the head-to-head helical conformation as well as for the double helical conformation of gramicidin A. The double-helical conformation was found to facilitate ion passage compared to the head-to-head helical dimer.     

Interaction of Escherichia coli heat-stable enterotoxin B with rat intestinal epithelial cells and membrane lipids

The binding of 125I-labeled Escherichia coli heat-stable enterotoxin B to rat intestinal epithelial cells was unsaturable and nonspecific, at concentrations well above that required to mediate biological events. Following its interaction with intestinal cells, approximately 50-80% of heat-stable enterotoxin B remained stably associated with the cells, implying that it was partitioned into the membrane and/or internalized by the cell. The toxin bound with different affinities to lipids isolated from intestinal epithelial cells, phospholipids, glycolipids, neutral lipids and to model membrane vesicles containing negatively charged lipids. These results indicate that heat-stable enterotoxin B utilizes the membrane bilayer, rather than a surface protein or glycoprotein in modulating toxin-induced enterotoxicity.     

Cell-detaching Escherichia coli (CDEC) strains from children with diarrhea: Identification of a protein with toxigenic activity

Twenty-four strains of cell-detaching Escherichia coli (CDEC) isolated from stool specimens in different cities in Brazil were examined for virulence properties. Aerobactin production and multiple antibiotic resistance were observed in most of the isolates. In hybridization studies, the alphahly, pap, and cnf sequences, common properties of this category of E. coli, were found in a minority of isolates. Half of the CDEC isolates had enteroaggregative DNA sequences (pet, astA, aggA), six strains carried the shet1 gene, nine strains carried the daaC sequence, and one strain carried the stp gene. Thirteen strains induced fluid accumulation in the rabbit intestinal loop assay. Supernatant filtrate of one of those strains, which did not hybridize with any of the toxin probes tested, induced destructive lesions in the rabbit ileal loop and enterotoxic activity in the Ussing chamber. A 12-kDa protein purified by 60% ammonium sulfate precipitation of the supernatant filtrate demonstrated a toxigenic effect that was inhibited by the anti-12-kDa protein antiserum.     

大肠杆菌异源表达的indigoidine与靛蓝的稳定性比较

【背景】Indigoidine是一种来源于微生物的无毒天然蓝色素。【目的】比较Indigoidine和靛蓝的色素稳定性,进而评价Indigoidine的色素稳定性。【方法】构建重组菌株Escherichia coli DH5α/p28s异源表达Indigoidine,考察可见光、紫外线、pH、温度、氧化还原剂、食品添加剂和金属离子对其与商品级靛蓝色素稳定性的影响。【结果】以N,N-二甲基甲酰胺为溶剂,Indigoidine和靛蓝都对可见光、紫外线敏感;2种色素在pH1.0-11.0时稳定,强碱性pH对色素破坏作用很大;Indigoidine抗Vc还原能力强于靛蓝,氧化剂可不同程度地降低2种色素的保存率;2种色素热稳定性不佳,在75℃以下时Indigoidine的色素稳定性优于靛蓝;食品添加剂中的柠檬酸和苯甲酸分别对Indigoidine和靛蓝均具有显著的护色效果;对这2种色素,Ca²⁺、Mg²⁺均具有护色效果,Na⁺、K⁺、Li⁺总体上没有明显的破坏作用,而Zn²⁺、Al³⁺、Cu²⁺、Fe²⁺、Fe³⁺则具有显著的破坏作用。【结论】Indigoidine色素稳定性明显优于靛蓝,具有广阔的开发应用前景。     

Staphylococcal alpha-toxin,streptolysin-O,and Escherichia coli hemolysin: prototypes of pore-forming bacterial cytolysins

Staphylococcal alpha-toxin, streptolysin-O, and Escherichia coli hemolysin are well-studied prototypes of pore-forming bacterial cytotoxins. Each is produced as a water-soluble single-chain polypeptide that inserts into target membranes to form aqueous transmembrane pores. This review will compare properties of the three toxin prototypes, highlighting the similarities and also the differences in their structure, mode of binding, mechanism of pore formation, and the responses they elicit in target cells. Pore-forming toxins represent the most potent and versatile weapons with which invading microbes damage the host macroorganism. Received: 31 July 1995 / Accepted: 3 November 1995     

Cytotoxic effect of multinucleation in HeLa cell cultures associated with the presence of Vir plasmid in Escherichia coli strains

The occurrence activity and localization of calmodulin in three heterocystous cyanobacteria of the genus Anabaena were studied. Boiled crude extracts caused a Ca2+-dependent stimulation of NAD kinase. Such a stimulation was blocked by EGTA and chlorpromazine, SDS-PAGE and Western blot analysis using antiserum against eukaryotic spinach calmodulin, revealed a polypeptide of about 17 kDa. Immunogold localization of calmodulin gave a dense gold label in both vegetative cells and heterocysts. The label was mainly confined to the centroplasm in vegetative cells, while it was evenly distributed in the cytoplasm of mature heterocysts.     

Ion channel formation by N-terminal domain: a common feature of OprFs of Pseudomonas and OmpA of Escherichia coli

The proteolytic fragments of OprFs of Pseudomonas aeruginosa and Pseudomonas fluorescens were identified, respectively, as the first 175 and 177 amino acids from the N-terminal domain. They induced ion channels after reincorporation into planar lipid bilayers (85 and 75 pS, respectively, in 1 M NaCl). A similar conductance value (72 pS) was found for the eight beta-strand OmpA N-terminal domain (OmpA171) of Escherichia coli. We conclude that the N-terminal domain of OprFs is sufficient to induce ion channels and the comparison with OmpA171, provides strong evidence of the existence of an eight-stranded beta-barrel in the N-terminal domain of OprFs.     

大肠杆菌染色体上严谨型启动子的构建

【背景】启动子的渗漏表达是代谢工程和合成生物学较为关注的问题,探索严谨型启动子使之能像开关一样控制基因的表达有助于解决这一问题。【目的】为避免在质粒上研究启动子带来的弊端,本研究将在染色体上对严谨型启动子进行构建和评价。【方法】基于4种调控元件四环素tetO、乳糖lacO、阿拉伯糖araC和鼠李糖rhaR的序列,以及2种来源的启动子PL和Plac序列,设计和组合构建了6个启动子PtetO2、PtetO3、PlacO2、PlacO3、PlacO+ara和PlacO+rha。应用CRISPR/Cas9系统将这6个启动子序列整合到大肠杆菌ATCC 8739染色体上,利用绿色荧光蛋白(Green fluorescent protein,GFP)的表达,分析这6个启动子的相对表达强度和严谨型控制情况。【结果】GFP表达分析显示,启动子PlacO+rha为最佳严谨型启动子,在无诱导剂时表达为0.02,有诱导剂时最大表达强度为lac Z基因启动子的12倍,相对控制范围为600倍。【结论】研究结果将为代谢工程和合成生物学中的精确调控基因表达奠定良好的应用基础。     

华东地区致初生仔猪腹泻大肠杆菌的O血清型和毒力因子

从江苏、江西、安徽等7个省疑似黄、白痢直肠棉拭及病死猪的十二指肠和肠系膜淋巴结中分离鉴定出339株病原性大肠杆菌。经O血清型鉴定,除77株未能定型、41株自凝外,测定出221个分离株的O血清型,这些分离株覆盖了64个血清型,以O107、O101、O20、O93、O11和O149为主,共99株,占定型菌株的44.80%。这些血清型与已报道的常见血清型间存在一定差异。运用黏附素单抗对以上菌株进行F4、F5、F6、F18、F41　5种黏附素检测,共97个分离株表达黏附素(28061%),而表达两种和3种黏附素的菌株分别有22株和8株,它们分别占表达黏附素菌株的22.68%和8.25%,其中单独表达F4、F6、F5+F41黏附素菌株分别有18、30、15株,分别占表达黏附素菌株的18.56%、30.93%和15.46%;同时运用多重PCR对其中145个分离株进行毒素基因(Sta、STb、LT、SLT2e)的检测,拥有Sta和STb毒素基因的菌株分别占检测菌株的51.72%和3724%。F6、F4、F5+F41和Sta、STb为该地区致初生仔猪腹泻大肠杆菌常见的毒力因子。     

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。     

EnvC, a new lipoprotein of the cytoplasmic membrane of Escherichia coli

Abstract A gene product with an apparent molecular mass of approximately 39000 Da can be identified in the cytoplasmic membrane of Escherichia coli upon expression of cloned envC . In this communication we report that the product was labelled with [³H]glycerol and [³H]palmitic acid, and a precursor molecule of increased molecular mass was accumulated when cells were treated with globomycin, a specific inhibitor for the prolipoprotein signal peptidase. The same precursor molecule was encoded by an envC mutant gene, in which the cysteine residue in a pentapeptide sequence, Leu-Ile-Ala-Gly-Cys²⁴ within the amino terminal region of EnvC, was replaced by tryptophane (Trp²⁴). This protein was not labelled with [³H]glycerol. The results demonstrate that the envC gene product represents a new lipoprotein of the cytoplasmic membrane of E. coli .