Salmonella typhi Ty2 OmpC Porin Induces Bactericidal Activity on U937 Monocytes

The immunogenic effect of Salmonella typhi OmpC porin during typhoid fever in humans was evaluated in vitro. Peripheral blood mononuclear cells from 17 patients were challenged with outer membrane preparations from Escherichia coli UH302 and UH302/pSTP2K2 strains, both lacking E. coli OmpF and OmpC porins, although UH302/pSTP2K2 expressed a plasmid-encoded S. typhi Ty2 OmpC. The mononuclear cell supernatants, immunized in vitro with OmpC antigen, derived from 10 out of 17 patients activated U937 bactericidal capacity. In contrast, the supernatants from the immunization with outer membrane preparation lacking S. typhi Ty2 OmpC induced a significantly reduced bactericidal capacity of U937 cells. This procedure should prove useful for in vitro characterization of cellular immunogens from exclusive human pathogens.     

Engineering the Escherichia coli outer membrane protein OmpC for metal bioadsorption

The outer membrane protein, OmpC, from Escherichia coli was used to display metal-binding poly-histidine peptides on the surface of this bacterium. SDS-PAGE analysis of outer membrane protein preparations confirmed the expression of the metal-binding epitopes inserted in position 162 of the mature OmpC protein. Display of these epitopes was confirmed by epifluorescence microscopy of cells bound to Ni²⁺-NTA-agarose beads and metal adsorption experiments. The cells harboring one or two copies of the metal binding epitope were able to adsorb 3 to 6 times more Zn²⁺ (13.8 mol g^–1 cell), Fe³⁺ (35.3 mol g^–1 cell), and Ni²⁺ (9.9 mol g^–1 cell) metallic ions than control cells expressing the wild-type OmpC.     

Overexpression of alginate lyase of <Emphasis Type="Italic">Pseudoalteromonas elyakovii</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>, purification,and characterization of the recombinant alginate lyase

The alyPEEC gene encoding alginate lyase from marine bacterium Pseudoalteromonas elyakovii IAM 14594 was subcloned into pBAD24 with arabinose promoter and sequenced, and overexpressed in TOP10 strain of E. coli after arabinose induction. Expression levels of alyPEEC gene in E. coli cells were over 39.6-fold higher than those in P. elyakovii IAM 14594 cells. The molecular mass of purified alginate lyase from the engineered E. coli cells was estimated to be 32.0 kDa. Optimum pH and temperature of the alginate lyase activity were 7.0 and 30 °C, respectively. The enzyme was unstable on heating and in acidic and alkaline solution. The enzyme activity was stimulated by the MgCl₂, NaCl, KCl, CaCl₂, BaCl₂ and MnCl₂, but was inhibited by the addition of 1.0 mM of EGTA, EDTA, SDS, ZnSO₄, AgNO₃, and CoCl₂. All the alginate, polyM and polyG could be converted into oligosaccharides with more than tetrasaccharides by the purified recombinant alginate lyase, suggesting that the recombinant alginate lyase produced by the engineered E. coli has highly potential application in seaweed genetics, food and pharmaceutical industries.     

Exchange of colicin receptor capacity between strains of Escherichia coli sensitive or resistant to colicin K-K235

Phage and colicin-resistant mutants were derived from Escherichia coli K-12P678. Two classes of phage T6 and colicin K-resistant mutants (genotype tsx) were isolated. Tsx-2 mutants, which demonstrated mucoid growth and increased sensitivities to many antibiotics, became sensitive to colicin K when pretreated with ethylenediaminetetraacetate (EDTA), whereas Tsx-1 mutants did not. Reassociation of EDTA-released material partially restored resistance to colicin K for Tsx-2 mutants. When EDTA-released material from strain P678 was associated with either class of K-resistant mutant, an increase in colicin K sensitivity resulted. Observations suggest that colicin K can act on its target site once it penetrates the cell surface. In addition, results suggest that functional colicin K receptors can be transferred from sensitive to resistant strains, thus conferring colicin sensitivity.Non-standard Abbreviations SDS sodium dodecyl sulfate     

Display of Polyhistidine Peptides on the Escherichia coli Cell Surface by Using Outer Membrane Protein C as an Anchoring Motif

A novel cell surface display system was developed by employing Escherichia coli outer membrane protein C (OmpC) as an anchoring motif. Polyhistidine peptides consisting of up to 162 amino acids could be successfully displayed on the seventh exposed loop of OmpC. Recombinant cells displaying polyhistidine could adsorb up to 32.0 μmol of Cd²⁺ per g (dry weight) of cells.     

Adherence Patterns and DNA Probe Types of Escherichia coli Isolated from Diarrheal Patients in China

One hundred and seventy-two strains of Escherichia coli isolated from diarrheal patients in Beijing, P. R. China, were analyzed for plasmid DNA profile, HEp-2 cell adherence ability and reactivity to 10 previously described DNA probes. They had not been recognized as pathogenic E. coli in China. Of the 110 strains tested, 76 (69%) contained one or multiple large plasmids. Of the 71 strains with the large plasmids 64 could adhere to HEp-2 cells. Of the 172 strains, 102 (59.3%) were hybridized with at least one of the 10 probes. Of those, seven strains hybridized with enteroaggregative E. coli (EAggEC) probe. Their serotypes were O128 (two strains), O6 (one strain), and O111 (one strain). Three strains were untypable. Six and three strains were hybridized with enteropathogenic E. coli (EPEC) attaching and effacing genes (eae) or EPEC adherence factor (EAF) probe, respectively. Two non-O157: H7 strains hybridized with enterohemorrhagic E. coli (EHEC) probe. Seventy-two strains (41.9%) hybridized with shiga-like toxin 2 or 1 (SLT2 or SLT1) probes. Among the SLT1 or SLT2 probe-positive strains, 54 hybridized with invasive (INV) plasmid probe developed for identification of enteroinvasive E. coli (EIEC) and Shigella species. The INV and SLT probe-positive strains might represent a new variety of verotoxin-producing E. coli (VTEC).     

Time-course determination of plasmid content in eukaryotic and prokaryotic cells using Real-Time PCR

A Real-Time PCR method was developed to monitor the plasmid copy number (PCN) in Escherichia coli and Chinese hamster ovary (CHO) cells. E. coli was transformed with plasmids containing a ColE1 or p15A origin of replication and CHO cells were transfected with a ColE1 derived plasmid used in DNA vaccination and carrying the green fluorescent protein (GFP) reporter gene. The procedure requires neither specific cell lysis nor DNA purification and can be performed in <30 min with dynamic ranges covering 0.9 pg–55 ng, and 5.0 pg–2.5 ng of plasmid DNA (pDNA) for E. coli and CHO cells, respectively. Analysis of PCN in E. coli batch cultures revealed that the maximum copy number per cell is attained in mid-exponential phase and that this number decreases on average 80% towards the end of cultivation for both types of plasmids. The plasmid content of CHO cells determined 24 h post-transfection was around 3 × 10⁴ copies per cell although only 37% of the cells expressed GFP one day after transfection. The half-life of pDNA was 20 h and around 100 copies/cell were still detected 6 days after transfection.     

Characterization of a phage specific to hemorrhagicEscherichia coli O157:H7 and disclosure of variations in host outer membrane protein OmpC

Phage AR1, previously known to infectEscherichia coli O157:H7 with high specificity, was further characterized for its genetic properties. The phage DNA sequences including capsid genes and a putative -glucosyltransferase gene(-gt) have been deduced. These sequences are conservative but not identical to those of T4 phage. However, a nonessential gene,SegD, organized within the capsid gene cluster of T4 is missing in the corresponding region of AR1 genome, and this characteristic has not been observed among T-even related phages. The difference between AR1 and T4 was further exemplified by their distinct host ranges. Strains ofE. coli O157:H7 collected from different sources were permissive to AR1 but resistant to T4 that normally infects K-12 strains ofE. coli through contact with the outer membrane protein OmpC. Thus, the O157:H7 strains must have a varied OmpC. Indeed, the OmpC sequence of O157:H7 strains was proved to differ from that of K-12 strains by a total of 15 amino acid substitutions and two gaps (a five-residue deletion and a four-residue insertion). The OmpC molecules are relatively conserved across the gram-negative bacteria, and this is the first time OmpC divergence has been found within the sameE. coli species. Since OmpC is located in the outer membrane and its expression is regulated by environmental conditions, alteration of the structure in pathogenic O157:H7 strains may have biological significance.     

Hemolytic activity of Serratia marcescens

A cell-bound hemolytic activity was found in several strains of Serratia marcescens. One Serratia cell per ten erythrocytes was sufficient to cause complete lysis of human erythrocytes within 2 h in the liquid assay. The hemolytic activity resided in the membrane fraction and could be inactivated by incubating cells with proteases. The hemolytic activity was greatly enhanced in actively metabolizing Serratia cells and was partially controlled by the iron supply. Hemolysis was accompanied by degradation of erythrocyte membrane proteins (band 3 and 6, glycophorin) and was independent of the blood group. The exoprotease secreted by S. marcescens in large amounts was not involved in hemolysis. Comparison with various hemolytic strains of Escherichia coli showed that hemolysis of erythrocytes was more pronounced with S. marcescens than with E. coli. In contrast to hemolysis by E. coli, lysis of erythrocytes by S. marcescens was not enhanced by Ca²⁺ ions.Dedicated to Professor Dr. Gerhart Drews on the occasion of his 60th birthday     

Ectopic Gene Conversions in Four <Emphasis Type="BoldItalic">Escherichia coli</Emphasis> Genomes: Increased Recombination in Pathogenic Strains

We characterized the ectopic gene conversions in the genomes of the K-12 MG1655, O157:H7 Sakai, O157:H7 EDL933, and CFT073 strains of E coli. Compared to the three pathogenic strains, the K-12 strain has a much smaller number of gene families, its gene families contain fewer genes, and gene conversions are less frequent. Whereas the three pathogenic strains have gene conversions covering hundreds of nucleotides when their flanking regions have as little as 50% similarity, flanking region similarity of at least 94% on both sides of the converted region is required to observe conversions of more than 87 nucleotides in the K-12 strain. Recombination is therefore more frequent and requires less sequence similarity in the three pathogenic strains than in K-12. This higher recombination level might be due to mutations in some of their mismatch-repair genes. In contrast with the gene conversions present in the yeast genome, the gene conversions found in the E. coli genomes do not occur more frequently between duplicated genes that are close to one another than between duplicated genes that are far apart and are randomly distributed along the length of the genes. In E. coli, gene conversions are not more frequent near the origin of replication. However, they do occur more frequently near the terminus of replication of the Sakai genome, where multigene family members are more abundant. This suggests that, in E. coli, gene conversions occur randomly between genes located in different chromosomal locations or located on different copies of the multiple chromosomes found in E. coli cells.     

Calcium Chloride Made E. coli Competent for Uptake of Extraneous DNA Through Overproduction of OmpC Protein

In the standard method of transformation of Escherichia coli with extraneous DNA, cells are made competent for DNA uptake by incubating in ice-cold 100?mM CaCl₂. Analysis of the whole protein profile of CaCl₂-treated E. coli cells by the techniques of one- and two-dimensional gel electrophoresis, MALDI-MS and immunoprecipitation revealed overproduction of outer membrane proteins OmpC, OmpA and heat-shock protein GroEL. In parity, transformation efficiency of E. coli ompC mutant by plasmid pUC19 DNA was found to be about 40?% lower than that of the wild type strain. Moreover, in E. coli cells containing groEL-bearing plasmid, induction of GroEL caused simultaneous overproduction of OmpC. On the other hand, less OmpC was synthesized in E. coli groEL mutant compared to its wild type counterpart, by CaCl₂-shock. From these results it can be suggested that in the process of CaCl₂-mediated generation of competence, the heat-shock chaperone GroEL has specific role in DNA entry into the cell, possibly through the overproduced OmpC and OmpA porins.     

Expression study with the Escherichia coli lep gene for leader peptidase in phototrophic purple bacteria

Synthesis and assembly of leader peptidase of Escherichia coli (signal peptidase I), was studied by heterologous expression of its lep gene in three species of phototrophic purple bacteria. Cell extracts of the recipient species showed neither cross reaction with antibodies against E. coli leader peptidase nor cleavage of the model substrate M13-procoat in vitro. The lep gene was transferred via conjugation using the plasmid expression vector for phototrophic bacteria pJAJ9. Plasmidborne leader peptidase enzyme was identified by immunochemical means. However, extracts of transconjugant cells showed no cleavage function. Trypsin digestion studies revealed that the enzyme was not properly integrated across the host membranes. The data suggest that cleaving enzymes for protein export and/or their assembly pathway in purple bacteria differ from the E. coli type.Abbreviations DMSO dimethylsulfoxide - EDTA ethylenediamine tetraacetic acid - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecyl sulfate     

鸡源大肠杆菌毒力基因检测及分子流行特征

[目的] 本试验旨在阐明鸡源大肠杆菌致病性及分子流行特性,为探索大肠杆菌流行途径制定合理的防控策略提供新思路。[方法] 2018–2019年在河北省采集病死鸡肝脏样品,通过选择培养基筛选、生化鉴定、血清凝集试验对分离菌株进行系统鉴定,应用PCR方法检测分离株中毒力基因流行情况。参考系统发育群分类方法对大肠杆菌进行分群分析,并参照McMLST网站数据库提供的7对管家基因序列进行多位点序列分型（multilocus sequence typing,MLST）分析。[结果] 结果显示,56株分离株符合大肠杆菌生化特征,分为8个生化表型,B4（30.36%）、B5（25%）和B2（23.21%）为主要生化表型。56株分离株大肠杆菌血清凝集试验均呈阳性,分为11种血清型,O₇₈（26.79%）、O₂（23.21%）、O₁₅₇（17.86%）和O₁（14.29%）为主要流行血清型。56株大肠杆菌共检测出15种肠外大肠杆菌毒力基因,未检出papC、ibeA和ibeB基因。黏附相关基因fimC和抗血清存活因子相关基因ompA携带率为100%。aatA、yijP、irp2、mat、iss,检出率分别为98.21%、98.21%、98.21%、96.43%、92.86%。同时,大肠杆菌与铁转运相关基因iroN、fyuA、iucD、irp2检出率均在80%以上。56株大肠杆菌中有20株属于肠出血型大肠杆菌（enterohemorrhagic E.coli,EHEC）,其次是肠聚集型大肠杆菌（enteroaggregative E.coli,EAEC）（n=4）、肠产毒素型大肠杆菌（Enterotoxigenic E.coli,ETEC）（n=2）。这些菌株D群分离株较多,其次是B2群。通过MLST分型分析,共分为22个ST型,其中ST88（n=7）、ST85（n=6）、ST243（n=6）型为主要流行型。[结论] 结果显示大肠杆菌血清型多样,毒力因子种类繁多,致病性大肠杆菌同时携带多种毒力基因,表明动物源大肠杆菌具有较强的毒力基础。     

Expression of subunits a and c of the sodium-dependent ATPase of Propionigenium modestum in Escherichia coli

The aim of the present study was to construct functional hybrid ATPases consisting of all Escherichia coli ATPase subunits excepts the F₀ subunits a or c which were replaced by the respective subunits of the Propionigenium modestum ATPase. This would give valuable information on the subunit(s) conferring the coupling ion specificity. Plasmids were constructed that carried the gene for subunit c (uncE) or subunit a (uncB) behind a tac promoter. These plasmids were transformed into E. coli strains which differed with respect to the unc operon and the expression of the P. modestum genes was verified biochemically. Enhanced expression of the P. modestum genes led to strong growth inhibition of all E. coli strains tested. However, the expressed P. modestum proteins could not functionally complement E. coli strains that lacked the homologous subunit.Abbreviations PCR Polymerase chain reaction - ACMA 9-amino-6-chloro-2-methoxyacridine - SDS sodium dodecyl sulfate - DCCD N,N-dicyclohexylcarbodiimide - PMSF pnenylmethyl sulfornyl fluoride - DFP dirsopropylfluorphosphat - TCA trichloroacetic acid     

Escherichia coli-induced productions of pro-inflammatory cytokines are regulated by MAP kinases and G-protein but not by Akt: Relationship with phylogenetic groups and resistance patterns

Introduction

We investigated the role of PI3-K, MAP kinases, and heterotrimeric G proteins in inducing cytokines production in human whole blood cultures stimulated by viable Escherichia coli (E. coli) clinical strains.

Materials and methods

We used eight E. coli strains that belong to different phylogenetic groups and presented by different antibiotic resistance patterns. Whole blood from healthy volunteers was incubated at 37 °C for 150 min, with lipopolysaccharide (LPS) from E. coli O111:B4 or selected viable E. coli clinical strains, with or without SB202190 (p38 inhibitor), PD98059 (ERK inhibitor), PTX (pertussis toxin; heterotrimeric G proteins inhibitor), wortmaninn (PI3-K inhibitor). The TNF-α, IL-1β, IL-10 and IFN-γ concentrations were measured in culture supernatants (ELISA).

Results

IL-10 and IFN-γ were not detectable. Susceptible strains induced higher TNF-α and IL-1β productions than β-lactam resistant strains (p < 0.05), with no difference between phylogenetic groups. A transformed strain carrying a plasmid-mediated AmpC-β-lactamase gene (CMY-2) induced lower TNF-α and IL-1β production than the parent wild type strain (p < 0.05). SB202190 (p38 inhibitor) and PD98059 (ERK inhibitor) reduced TNF-α concentrations by, respectively, 80% (p < 0.05) and 50% (p < 0.05). Wortmaninn (PI3-K inhibitor) had no significant effect. PTX (heterotrimeric G proteins inhibitor) altered TNF-α production after viable bacteria stimulation (1.7-fold increase; p < 0.05) but not after LPS (TLR-4) stimulation. Regarding IL-1β, wortmaninn, SB202190 and PTX had no significant effect whereas PD98059 significantly decreased production in whole cell cultures (p < 0.05).

Conclusion

Susceptible strains induce greater TNF-α and IL-1β productions than resistant strains. ERK kinase plays a major role in viable E. coli strains inducing TNF-α and IL-1β production. E. coli exerts an effect on the pertussis toxin-sensitive G-protein through a TLR-4-independent mechanism.     

Roles of mitogen-activated protein kinase pathways during <Emphasis Type="Italic">Escherichia coli-</Emphasis>induced apoptosis in U937 cells

Escherichia coli (E. coli) infections play an important and growing role in the clinic. In the present study, we investigated the involvement of members of the mitogen-activated protein kinase (MAPK) superfamily, including extracellular signal-regulated kinase (ERK), c-Jun NH₂-terminal kinase (JNK) and p38 MAPK, and caspase-3 and 9 activity in E. coli-induced apoptosis in human U937 cells. We found that E. coli induces apoptosis in U937 cell lines in a dose- and time-dependent manner, p38 MAPK and JNK were activated after 10 min of infection with E. coli. In contrast, ERK1/2 was down-regulated in a time-dependent manner. The levels of total (phosphorylation state-independent) p38 MAPK, JNK and ERK1/2 did not change in E. coli-infected U937 cells at all times examined. Moreover, exposure of U937 cells to E. coli led to caspase-3 and 9 activity. For the evaluation of the role of MAPKs, PD98059, SB203580 and SP600125 were used as MAPKs inhibitors for ERK1/2, p38 MAPK and JNK. Inhibition of ERK1/2 with PD98059 caused further enhancement in apoptosis and caspase-3 and 9 activity, while a selective p38 MAPK inhibitor, SB203580 and JNK inhibitor, SP600125 significantly inhibited E. coli-induced apoptosis and caspase-3 and 9 activity in U937 cells. The results were further confirmed by the observation that the caspase inhibitors Z-DEVD-FMK and Z-LEHD-FMK blocked E. coli-induced U937 apoptosis. Taken together, we have shown that E. coli increase p38 MAPK and JNK and decrease ERK1/2 phosphorylation and increase caspase-3 and 9 activity in U937 cells.     

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.     

Structure‐kinetic relationship of carbapenem antibacterials permeating through E. coli OmpC porin

The emergence of Gram‐negative “superbugs” exhibiting resistance to known antibacterials poses a major public health concern. Low molecular weight Gram‐negative antibacterials are believed to penetrate the outer bacterial membrane (OM) through porin channels. Therefore, intracellular exposure needed to drive antibacterial target occupancy should depend critically on the translocation rates through these proteins and avoidance of efflux pumps. We used electrophysiology to study the structure‐translocation kinetics relationships of a set of carbapenem antibacterials through purified porin OmpC reconstituted in phospholipid bilayers. We also studied the relative susceptibility of OmpC+ and OmpC‐ E. coli to these compounds as an orthogonal test of translocation. Carbapenems exhibit good efficacy in OmpC‐expressing E. coli cells compared with other known antibacterials. Ertapenem, which contains an additional acidic group compared to other analogs, exhibits the fastest entry into OmpC (k_on ≈ 2 × 10⁴ M^?1 s^?1). Zwitterionic compounds with highly polar groups attached to the penem‐2 ring, including panipenem, imipenem and doripenem exhibit faster k_on (>10⁴ M^?1 s^?1), while meropenem and biapenem with fewer exposed polar groups exhibit slower k_on (～5 × 10³ M^?1 s^?1). Tebipenem pivoxil and razupenem exhibit ～13‐fold slower k_on (～1.5 × 10³ M^?1 s^?1) than ertapenem. Overall, our results suggest that (a) OmpC serves as an important route of entry of these antibacterials into E. coli cells; and (b) that the structure‐kinetic relationships of carbapenem translocation are governed by H‐bond acceptor/donor composition (in accordance with our previous findings that the enthalpic cost of transferring water from the constriction zone to bulk solvent increases in the presence of exposed nonpolar groups). Proteins 2014; 82:2998–3012. © 2014 Wiley Periodicals, Inc.     

Inactivation and injury of Escherichia coli O157:H7 and Staphylococcus aureus by pulsed electric fields

Two pathogenic microorganisms Escherichia coli O157:H7 and Staphylococcus aureus, suspended in peptone solution (0.1% w/v) were treated with 12, 14, 16 and 20 kV/cm electric field strengths with different pulse numbers up to 60 pulses. Pulsed electric field (PEF) treatment at 20 kV/cm with 60 pulses provided nearly 2 log reduction in viable cell counts of E. coli O157:H7 and S. aureus. S. aureus cells were slightly more resistant than E.coli O157:H7 cells. The results related to the effect of initial cell concentration of E. coli O157:H7 on the PEF inactivation showed that more inactivation was obtained by decreasing initial cell concentration. Any possible injury by PEF was also investigated after applying 20 kV/cm electric field to the microorganisms. As a result, it was determined that there was 35.92 to 43.36% injury in E. coli O157:H7 cells, and 17.26 to 30.86% injury in S. aureus cells depending on pulse number. The inactivation results were also described by a kinetic model.     

Bioadsorption of cadmium ion by cell surface-engineered yeasts displaying metallothionein and hexa-His

The Cd²⁺-chelating abilities of yeast metallothionein (YMT) and hexa-His displayed on the yeast-cell surface were compared. Display of YMT and hexa-His by -agglutinin-based cell-surface engineering was confirmed by immunofluorescent labeling. Surface-engineered yeast cells with YMT and hexa-His fused in tandem showed superior cell-surface adsorption and recovery of Cd²⁺ under EDTA treatment on the cell surface than hexa-His-displaying cells. YMT was demonstrated to be more effective than hexa-His for the adsorption of Cd²⁺. Yeast cells displaying YMT and/or hexa-His exhibited a higher potential for the adsorption of Cd²⁺ than Escherichia coli cells displaying these molecules. In order to investigate the effect of the displayed YMT and hexa-His on sensitivity to toxic Cd²⁺, growth in Cd²⁺-containing liquid medium was monitored. Unlike hexa-His-displaying cells, cells displaying YMT and hexa-His fused in tandem induced resistance to Cd²⁺ through active and enhanced adsorption of toxic Cd²⁺. These results indicate that YMT-displaying yeast cells are a unique bioadsorbent with a functional chelating ability superior to that of E. coli.