Occurrence and persistence of <Emphasis Type=Italic>Escherichia coli</Emphasis> O157:H7 in water

Escherichia coli O157:H7 has been associated with water related outbreaks. It has been isolated from surface and ground waters. It is capable of survival in water for days to weeks     

Cloning and expression of <Emphasis Type=Italic>Tenebrio molitor</Emphasis> antifreeze protein in <Emphasis Type=Italic>Escherichia coli</Emphasis>

A novel antifreeze protein cDNA was cloned by RT-PCR from the larva of the yellow mealworm Tenebrio molitor. The coding fragment of 339 bp encodes a protein of 112 amino acid residues and was fused to the expression vectors pET32a and pTWIN1. The resulted expression plasmids were transformed into Escherischia coli strains BL21 (DE3), ER2566, and Origami B (DE3), respectively. Several strategies were used for expression of the highly disulfide-bonded β-helix-contained protein with the activity of antifreeze in different expression systems. A protocol for production of refolded and active T. molitor antifreeze protein in bacteria was obtained.     

No direct binding of the heat-labile enterotoxin of <Emphasis Type=Italic>Escherichia coli</Emphasis> to <Emphasis Type=Italic>E. coli</Emphasis> lipopolysaccharides

A novel carbohydrate binding site recognizing blood group A and B determinants in a hybrid of cholera toxin and Escherichia coli heat-labile enterotoxin B-subunits (termed LCTBK) has previously been described, and also the native heat-labile enterotoxin bind to some extent to blood group A/B terminated glycoconjugates. The blood group antigen binding site is located at the interface of the B-subunits. Interestingly, the same area of the B-subunits has been proposed to be involved in binding of the heat-labile enterotoxin to lipopolysaccharides on the bacterial cell surface. Binding of the toxin to lipopolysaccharides does not affect the GM1 binding capacity. The present study aimed at characterizing the relationship between the blood group A/B antigen binding site and the lipopolysaccharide binding site. However, no binding of the B-subunits to E. coli lipopolysaccharides in microtiter wells or on thin-layer chromatograms was obtained. Incubation with lipopolysaccharides did not affect the binding of the B-subunits of heat-labile enterotoxin of human isolates to blood group A-carrying glycosphingolipids, indicating that the blood group antigen site is not involved in LPS binding. However, the saccharide competition experiments showed that GM1 binding reduced the affinity for blood group A determinants and vice versa, suggesting that a concurrent occupancy of the two binding sites does not occur. The latter finding is related to a connection between the blood group antigen binding site and the GM1 binding site through residues interacting with both ligands.     

A role of <Emphasis Type=Italic>ygfZ</Emphasis> in the <Emphasis Type=Italic>Escherichia coli</Emphasis> response to plumbagin challenge

Plumbagin is found in many herbal plants and inhibits the growth of various bacteria. Escherichia coli strains are relatively resistant to this drug. The mechanism of resistance is not clear. Previous findings showed that plumbagin treatment triggered up-regulation of many genes in E. coli including ahpC, mdaB, nfnB, nfo, sodA, yggX and ygfZ. By analyzing minimal inhibition concentration and inhibition zones of plumbagin in various gene-disruption mutants, ygfZ and sodA were found critical for the bacteria to resist plumbagin toxicity. We also found that the roles of YgfZ and SodA in detoxifying plumbagin are independent of each other. This is because of the fact that ectopically expressed SodA reduced the superoxide stress but not restore the resistance of bacteria when encountering plumbagin at the absence of ygfZ. On the other hand, an ectopically expressed YgfZ was unable to complement and failed to rescue the plumbagin resistance when sodA was perturbed. Furthermore, mutagenesis analysis showed that residue Cys228 within YgfZ fingerprint region was critical for the resistance of E. coli to plumbagin. By solvent extraction and HPLC analysis to follow the fate of the chemical, it was found that plumbagin vanished apparently from the culture of YgfZ-expressing E. coli. A less toxic form, methylated plumbagin, which may represent one of the YgfZ-dependent metabolites, was found in the culture supernatant of the wild type E. coli but not in the ΔygfZ mutant. Our results showed that the presence of ygfZ is not only critical for the E coli resistance to plumbagin but also facilitates the plumbagin degradation.     

Characterization of <Emphasis Type=Italic>Escherichia coli</Emphasis> EutD: a phosphotransacetylase of the ethanolamine operon

The Escherichia coli genes pta and eutD encode proteins containing the phosphate-acetyltransferase domain. EutD is composed only by this domain and belongs to the ethanolamine operon. This enzyme has not been characterized yet, and its relationship to the multimodular E. coli phosphotransacetylase (Pta) remains unclear. In the present work, a detailed characterization of EutD from E. coli (EcEutD) was performed. The enzyme is a more efficient phosphotransacetylase than E. coli Pta (EcPta) in catalyzing its reaction in either direction and assembles as a dimer, being differentially modulated by EcPta effectors. When comparing EutD and Pta, both from E. coli, certain divergent regions of the primary structure responsible for their unique properties can be found. The growth on acetate of the E. coli pta acs double-mutant strain, was complemented by either introducing EcEutD or by inducing the eut operon with ethanolamine. In this case, the expression of a phosphotransacetylase different from Pta was confirmed by activity assays. Overall, the results indicate that EcEutD and Pta, although able to catalyse the same reaction, display differential efficiency and regulation, and also differ in the induction of their expression. However, under certain growth conditions, they can fulfil equal roles in E. coli metabolism.     

Enhanced Soluble Expression of a Thermostable Cellulase from <Emphasis Type=Italic>Clostridium thermocellum</Emphasis> in <Emphasis Type=Italic>Escherichia coli</Emphasis>

In this study, the cellulase gene celD from Clostridium thermocellum was cloned into expression vectors pET-20b(+) and pHsh. While high expression can be achieved by means of both these expression systems, only the pHsh expression system gives soluble proteins. By weakening the mRNA secondary structure and replacing the rare codons for the N-terminal amino acids of the target protein, the expression level of CelD was increased from 4.1 ± 0.3 to 6.4 ± 0.4 U ml⁻¹ in LB medium. Recombinant CelD was purified by heat treatment followed by Ni–NTA affinity. The purified CelD exhibited the highest activity at pH 5.4 and 60°C, and retained more than 50% activity after incubation at 70°C for 1 h. The cellulase activity of CelD was significantly enhanced by Ca²⁺ but inhibited by EDTA. The favorable properties of CelD offer the potential for genetic modification of strains for biomass degradation. Presently, one of the major bottlenecks for industrial cellulase users is the high cost of enzyme production. The high level expression of soluble enzymes from the pHsh expression system offers a novel approach for the production of cellulases to be used in various agro-industrial processes such as chemical, food and textile.     

Phosphoenolpyruvate:glucose phosphotransferase system modification increases the conversion rate during <Emphasis Type=SmallCaps>l</Emphasis>-tryptophan production in <Emphasis Type=Italic>Escherichia coli</Emphasis>

Escherichia coli FB-04(pta1), a recombinant l-tryptophan production strain, was constructed in our laboratory. However, the conversion rate (l-tryptophan yield per glucose) of this strain is somewhat low. In this study, additional genes have been deleted in an effort to increase the conversion rate of E. coli FB-04(pta1). Initially, the pykF gene, which encodes pyruvate kinase I (PYKI), was inactivated to increase the accumulation of phosphoenolpyruvate, a key l-tryptophan precursor. The resulting strain, E. coli FB-04(pta1)ΔpykF, showed a slightly higher l-tryptophan yield and a higher conversion rate in fermentation processes. To further improve the conversion rate, the phosphoenolpyruvate:glucose phosphotransferase system (PTS) was disrupted by deleting the ptsH gene, which encodes the phosphocarrier protein (HPr). The levels of biomass, l-tryptophan yield, and conversion rate of this strain, E. coli FB-04(pta1)ΔpykF/ptsH, were especially low during fed-batch fermentation process, even though it achieved a significant increase in conversion rate during shake-flask fermentation. To resolve this issue, four HPr mutations (N12S, N12A, S46A, and S46N) were introduced into the genomic background of E. coli FB-04(pta1)ΔpykF/ptsH, respectively. Among them, the strain harboring the N12S mutation (E. coli FB-04(pta1)ΔpykF-ptsHN12S) showed a prominently increased conversion rate of 0.178 g g^?1 during fed-batch fermentation; an increase of 38.0% compared with parent strain E. coli FB-04(pta1). Thus, mutation of the genomic of ptsH gene provided an alternative method to weaken the PTS and improve the efficiency of carbon source utilization.     

Lactoferrin: an iron-binding antimicrobial protein against <Emphasis Type=Italic>Escherichia coli</Emphasis> infection

Escherichia coli (E. coli) are the most common aerobic gram-negative bacilli in a normal intestinal tract. They cause most of the intra-abdominal infections, wound infections associated with abdominal surgery, and septicemia. Most of these infections are of endogenous intestinal origin. Lactoferrin (LF) is an iron-binding glycoprotein found in milk and various external secretions. This protein has been found to have a number of biological functions, including antimicrobial, anti-cancer, antioxidant, and immunomodulatory effects. Partial degradation of LF by pepsin can give rise to peptides termed lactoferricin (LFcin) with more potent antimicrobial activity. LF and LFcin have been shown to inhibit the growth of a number of pathogenic bacteria (including E. coli and antibiotic-resistant strains), fungi, and even viruses in both in vitro and in vivo studies. We previously demonstrated that both recombinant porcine LF (pLF) produced from yeast and a synthetic 20-residue porcine LFcin peptide exhibit antimicrobial activity in vitro. In one of our recent studies, we performed pathogen challenges, including pathogenic E. coli, Staphylococcus aureus and Candida albicans, of the digestive tract of a transgenic milk-fed animal model. The results showed that LF has broad spectrum antimicrobial activity in the digestive tract and protects the mucosa of the small intestine from injury. Our following study also revealed that pLF as a feedstuff additive enhances avian immunity, including antibody formation and cell-mediated immunity. All of these results suggest that LF could be a novel natural protein in the treatment and prevention of infections with E. coli or antibiotic-resistant bacteria strains.     

Protective action of reactivating factor of <Emphasis Type=Italic>Luteococcus japonicus</Emphasis> subsp. <Emphasis Type=Italic>casei</Emphasis> toward cells of <Emphasis Type=Italic>Escherichia coli</Emphasis> reparation mutants inactivated with UV-light

Reactivating factor (RF) from Luteococcus japonicus subsp. casei had a protective action on UV-irradiated cells of Escherichia coli AB1157 with a native reparation system and on cells of isogenic reparation mutants of E. coli UvrA⁻, RecA⁻, and PolA⁻: the effect resulted in multifold increase of survivability. Defense action of L. casei exometabolite is not connected with stimulating reparation systems in E. coli, and, probably, it is mediated by involvement of the exometabolite in the mechanism of cell division. RF did not provoke the reactivation of E. coli cells inactivated by UV-light.     

PriA participates in nascent DNA synthesis in <Emphasis Type=Italic>Escherichia coli</Emphasis>

The question of whether discontinuous DNA replication operates only for the lagging strand or for both strands in E. coli remains unresolved. In this study, the participation of priA, B, C and rep genes in discontinuous DNA replication was examined by analyzing the size distribution of nascent DNA synthesized in wild-type, lig-7 and polA4113 genetic backgrounds. Inactivation of priA, but not priB, priC or rep, resulted in a significant increase of high molecular weight (HMW) DNA in the short pulse-labeled DNA in the wild-type lig ⁺ polA ⁺ strains. Inactivation of priA also produced a significant increase of HMW DNA in the nascent DNA synthesized in lig-7 and polA4113 strains. These results indicate that PriA is involved in the discontinuous synthesis of nascent DNA.     

Biosynthesis of fluorescent cyanobacterial allophycocyanin trimer in <Emphasis Type=Italic>Escherichia coli</Emphasis>

Allophycocyanin (APC), a cyanobacterial photosynthetic phycobiliprotein, functions in energy transfer as a light-harvesting protein. One of the prominent spectroscopic characteristics of APC is a strong red-shift in the absorption and emission maxima when monomers are assembled into a trimer. Previously, holo-APC α and β subunits (holo-ApcA and ApcB) were successfully synthesized in Escherichia coli. In this study, both holo-subunits from Synechocystis sp. PCC 6803 were co-expressed in E. coli, and found to self-assemble into trimers. The recombinant APC trimer was purified by metal affinity and size-exclusion chromatography, and had a native structure identical to native APC, as determined by characteristic spectroscopic measurements, fluorescence quantum yield, tryptic digestion analysis, and molecular weight measurements. Combined with results from a study in which only the monomer was formed, our results indicate that bilin synthesis and the subsequent attachment to apo-subunits are important for the successful assembly of APC trimers. This is the first study to report on the assembly of recombinant ApcA and ApcB into a trimer with native structure. Our study provides a promising method for producing better fluorescent tags, as well as a method to facilitate the genetic analysis of APC trimer assembly and biological function.     

Formation of glycated recombinant leghemoglobin in <Emphasis Type=Italic>Escherichia coli</Emphasis> cells

A nonenzymatic glycation of the recombinant leghemoglobin expressed in Escherichia coli cells was demonstrated for the first time. This process involved the heme pocket and gave low-spin leghemoglobin species. A correlation between the degree of E. coli protein glycation and synthesis of poly-β-hydroxybutyric acid was found, suggesting that the accumulation of reserve carbon sources and nonenzymatic glycation could be alternative processes.     

Mutation <Emphasis Type=Italic>clp</Emphasis>A::<Emphasis Type=Italic>kan</Emphasis> of the gene for an Hsp100 family chaperone impairs the DnaK-dependent refolding of proteins in <Emphasis Type=Italic>Escherichia coli</Emphasis>

The rate and level of DnaK-dependent refolding of heat-inactivated Vibrio fischeri luciferase in the clp A mutant (clp A:: kan) were considerably lower then in wild-type cells. The decline in refolding level progressed with increasing heat inactivation time. A mutation of clp P had no influence on the kinetics and level of luciferase refolding. Approximately equal amounts of the DnaKJE chaperone were synthesized upon heat shock induction in E. coli clp A + and E. coli clpA::kan cells. It was assumed that, like homologous chaperone ClpB, ClpA is involved in disaggregation of denatured proteins, increasing the refolding efficiency. This in vivo phenomenon occurred only upon a prolonged incubation of cells at a higher temperature, which led to the formation of large protein aggregates that were poorly refoldable by the DnaKJE system.     

Cellular and proteomic responses of <Emphasis Type=Italic>Escherichia coli</Emphasis> JK-17 exposed to the <Emphasis Type=Italic>Rosa hybrida</Emphasis> flower extract

The purpose of this work was to characterize the cellular and proteomic responses of Escherichia coli JK-17 exposed to the rose flower extract (Rosa hybrida). The bacterial isolate was enriched and isolated from contaminated food. 16S rRNA sequence analyses revealed that the strain was 99% similar to the E. coli species cluster; therefore, this strain was designated E. coli JK-17. The rose flower extract showed a dose-dependent antibacterial effect on E. coli JK-17. Treatment of E. coli JK-17 with 50 and 100 mg/mL of the rose flower extract completely inhibited growth within 12 and 6 h of incubation. The stress shock proteins (SSPs) were induced with different concentrations of rose flower extract. The proteins were identified as 70-kDa DnaK and 60-kDa GroEL by SDS-PAGE and Western blot using anti-DnaK and anti-GroEL monoclonal antibodies. The levels of SSPs induced by the rose flower extract increased when the exposure time to the rose flower extract was increased. SDS-PAGE with silver staining revealed that the amount of lipopolysaccharide (LPS) in E. coli JK-17 increased or decreased with different concentrations and exposure times of the rose flower extract. To identify proteins induced by the rose flower extract, 2-dimensional electrophoresis (2-DE) was applied to soluble protein fractions of E. coli JK-17 cultures. In the pH range of 4 ∼ 7, more than 250 spots were detected on the silver stained gels. Notably, 15 protein spots were increased or decreased after treatment with the rose flower extract. Twelve up-regulated proteins were identified as chaperones (DnaK and GroEL) and porin proteins (PhoE, RfaI, RfaG, MdoH, and WzzE) by MALDITOF mass spectrometry, and three down-regulated proteins were identified, including proteins involved in energy and DNA metabolism (SdhA and GyrB), and amino acid biosynthesis (GltK). Using scanning electron microscopic analysis, some cells were shown to adopt irregular rod shapes and wrinkled surfaces after treatment with the rose flower extract. These results provide clues for better understanding the mechanism of rose flower extract-induced stress and cytotoxicity in E. coli JK-17.     

Probiotic <Emphasis Type=Italic>Lactobacillus</Emphasis> strains: <Emphasis Type=Italic>in vitro</Emphasis> and <Emphasis Type=Italic>in vivo</Emphasis> studies

Three Lactobacillus strains (LOCK 0900, LOCK 0908, LOCK 0919) out of twenty-four isolates were selected according to their antagonistic activity against pathogenic bacteria, resistance to low pH and milieu of bile salts. Intragastric administration of a mixture of these strains to Balb/c mice affected cytokine T_H1-T_H2 balance toward nonallergic T_H1 response. Spleen cells, isolated from lactobacilli-treated mice and re-stimulated in vitro with the mixture of heat-inactivated tested strains, produced significantly higher amounts of anti-allergic tumor necrosis factor- and interferon-γ than control animals whereas the level of pro-allergic interleukin-5 was significantly lower. Lactobacillus cells did not translocate through the intestinal barrier into blood, liver and spleen; a few Lactobacillus cells found in mesenteric lymph nodes could create antigenic reservoir activating the immune system. The mixture of Lactobacillus LOCK 0900, LOCK 0908 and LOCK 0919 strains represents a probiotic bacterial preparation with possible use in prophylaxis and/or therapy of allergic diseases.