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.     

Enhanced production and characterization of a β-glucosidase from <Emphasis Type="Italic">Bacillus halodurans</Emphasis> expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A putative β-glucosidase gene from the genome of Bacillus halodurans C-125 was expressed in E. coli under the regulation of T7lac promoter. On induction with isopropyl-β-D-1-thiogalactopyranoside, the enzyme expressed at ∼40% of the cell protein producing 238 mg/liter culture. With increase in culture cell density to A ₆₀₀ 12 in auto-inducing M9NG medium, β-glucosidase production increased 3-fold. Approximately 70% of the expressed enzyme was in a soluble form, while the rest was in an insoluble fraction of the cell lysate. The soluble and active form of the expressed enzyme was purified by ammonium sulfate precipitation followed by ion-exchange chromatography to a purity >98%. The mass of the enzyme as determined by MALDI-TOF mass spectrometry was 51,601 Da, which is nearly the same as the calculated value. Phylogenetic analysis of the β-glucosidase of B. halodurans was found to cluster with members of the genus Bacillus. Temperature and pH optima of the enzyme were found to be 45°C and 8.0, respectively, under the assay conditions. K _m and k _cat against p-nitrophenyl-β-D-glucopyranoside were 4 mM and 0.75 sec⁻¹, respectively. To our knowledge, this is the first report of high-level expression and characterization of a β-glucosidase from B. halodurans.     

Evaluation of Petrifilm™ Select <Emphasis Type="Italic">E. coli</Emphasis> Count Plate medium to discriminate antimicrobial resistant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Screening and enumeration of antimicrobial resistant Escherichia coli directly from samples is needed to identify emerging resistant clones and obtain quantitative data for risk assessment. Aim of this study was to evaluate the performance of 3M™ Petrifilm™ Select E. coli Count Plate (SEC plate) supplemented with antimicrobials to discriminate antimicrobial-resistant and non-resistant E. coli.     

Enteropathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis> (EPEC) in Antarctic fur seals <Emphasis Type="Italic">Arctocephalus gazella</Emphasis>

Rectal swabs were collected from Antarctic fur seal pups Arctocephalus gazella at Cape Shirreff, South Shetland Islands, and analyzed for the presence of anthropogenic pathogens. Two of the 33 pups tested positive for enteropathogenic Escherichia coli (EPEC). These samples are the first records of EPEC in Antarctic wildlife and suggest that more needs to be done to protect the Antarctic fauna from exotic anthropogenic pathogens.     

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.     

DNA plasmid production in different host strains of <Emphasis Type="Italic">Escherichia coli</Emphasis>

We compared plasmid DNA production in 13 strains of Escherichia coli in shake flasks using media containing glucose or glycerol. DNA yield from either carbon source showed small correlation with maximum growth rate. Three strains, SCS1-L, BL21 and MC4100, were selected for a controlled exponential fed-batch process at a growth rate of 0.14 h⁻¹ to an optical density of about 70, followed by a four-hour heat treatment. Prior to heat treatment, SCS1-L generated 15.4 mg DNA/g, BL21 generated 11.0 mg DNA/g and MC4100 generated 7.9 mg DNA/g, while after heat treatment the strains attained DNA yields, respectively, of 18.0, 15.0 and 6.8 mg/g. The strains also varied in their percentage of supercoiled DNA after heat treatment, with SCS1-L averaging 66% supercoiled, BL21 17% and MC4100 40%. We further investigated the two strains that yielded the highest percentage of supercoiled DNA (SCS1-L and MC4100) at a higher growth rate of 0.28 h⁻¹. At this condition, a slightly lower DNA yield was generated faster, and the percentage of supercoiled DNA increased. Heat treatment improved DNA yield, and surprisingly did so to a greater extent at the higher growth rate. As a consequence of these factors, higher growth rates might be advantageous for DNA production.     

Translation initiation region sequence preferences in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

The mRNA translation initiation region (TIR) comprises the initiator codon, Shine-Dalgarno (SD) sequence and translational enhancers. Probably the most abundant class of enhancers contains A/U-rich sequences. We have tested the influence of SD sequence length and the presence of enhancers on the efficiency of translation initiation.     

The new pLAI (<Emphasis Type="Italic">lux</Emphasis> regulon based auto-inducible) expression system for recombinant protein production in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

After many years of intensive research, it is generally assumed that no universal expression system can exist for high-level production of a given recombinant protein. Among the different expression systems, the inducible systems are the most popular for their tight regulation. However, induction is in many cases less favorable due to the high cost and/or toxicity of inducers, incompatibilities with industrial scale-up or detrimental growth conditions. Expression systems using autoinduction (or self-induction) prove to be extremely versatile allowing growth and induction of recombinant proteins without the need to monitor cell density or add inducer. Unfortunately, almost all the actual auto inducible expression systems need endogenous or induced metabolic changes during the growth to trigger induction, both frequently linked to detrimental condition to cell growth. In this context, we use a simple modular approach for a cell density-based genetic regulation in order to assemble an autoinducible recombinant protein expression system in E. coli.     

Deletion of methylglyoxal synthase gene (<Emphasis Type="Italic">mgsA</Emphasis>) increased sugar co-metabolism in ethanol-producing <Emphasis Type="Italic">Escherichia coli</Emphasis>

The use of lignocellulose as a source of sugars for bioproducts requires the development of biocatalysts that maximize product yields by fermenting mixtures of hexose and pentose sugars to completion. In this study, we implicate mgsA encoding methylglyoxal synthase (and methylglyoxal) in the modulation of sugar metabolism. Deletion of this gene (strain LY168) resulted in the co-metabolism of glucose and xylose, and accelerated the metabolism of a 5-sugar mixture (mannose, glucose, arabinose, xylose and galactose) to ethanol.     

<Emphasis Type="Italic">Bacillus anthracis</Emphasis>, <Emphasis Type="Italic">Francisella tularensis</Emphasis> and <Emphasis Type="Italic">Yersinia pestis</Emphasis>. The most important bacterial warfare agents — <Emphasis Type="Italic">review</Emphasis>

There are three most important bacterial causative agents of serious infections that could be misused for warfare purposes: Bacillus anthracis (the causative agent of anthrax) is the most frequently mentioned one; however, Fracisella tularensis (causing tularemia) and Yersinia pestis (the causative agent of plague) are further bacterial agents enlisted by Centers for Disease Control and Prevention into the category A of potential biological weapons. This review intends to summarize basic information about these bacterial agents. Military aspects of their pathogenesis and the detection techniques suitable for field use are discussed.     

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.     

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.     

Sensitivity of various <Emphasis Type="Italic">Escherichia coli</Emphasis> strains to 2,4,6-trinitrotoluene

The sensitivity of Escherichia coli strains K-12 and 055 to 2,4,6-trinitrotoluene (TNT) was found to correlate with the structural and functional properties of the outer lipoprotein membrane. The protective ability of the membrane of strain 055 is much lower than that of K-12. This is the cause of the greater sensitivity of 055 to the toxic action of TNT. High TNT concentrations (100–200 mg/l) suppressed the growth of 055, whereas K-12 grew at all TNT concentrations studied. Both strains adapted to high TNT concentrations by converting it by either nitroreduction or denitritation depending on concentration. The denitritation system of strain 055 started TNT degradation earlier than that of K-12.Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 1, 2005, pp. 53–57.Original Russian Text Copyright © 2005 by Kurinenko, Denivarova, Yakovleva.     

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.     

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.     

Interaction of <Emphasis Type="Italic">Bdellovibrio bacteriovorus</Emphasis> with bacteria <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> and <Emphasis Type="Italic">Helicobacter pylori</Emphasis>

Interaction of Bdellovibrio bacteriovorus 100NCJB with bacteria Campylobacter jejuni (strains 1, 2, 3, 4, and 5) and Helicobacter pylori, strain TX30a, was confirmed. The results indicate that lytic activity of bdellovibrios both in liquid media and cells attached to a surface was observed. The potential use of the antimicrobial activity of predatory bacteria for environmental bioprotection and public health is discussed.     

Multiple host-ant use by the predatory social parasite <Emphasis Type="Italic">Phengaris</Emphasis> (=<Emphasis Type="Italic">Maculinea</Emphasis>) <Emphasis Type="Italic">arion</Emphasis> (Lepidoptera,Lycaenidae)

Phengaris (=Maculinea) arion is an endangered social parasite of Myrmica ants, and for a very long time was considered as specific to Myrmica sabuleti. Previous studies carried out in Poland suggested some discrepancies within this assumption, and therefore a much more intensive survey was undertaken. The host ant use of P. arion was studied at five sites in different types of biotopes in Poland, i.e. xerothermal grasslands where Thymus pulegioides was used as a larval food plant by the butterfly, and more or less sandy biotopes with Thymus serpyllum. Altogether nine Myrmica species were recorded, and considerable variation in species composition and density of nests was recorded. At four localities M. sabuleti proved to be the most common ant. A total of 529 Myrmica nests were examined, and only 20 of them contained larvae and pupae of P. arion. Host ants belonged to five different species, i.e. M. sabuleti, Myrmica scabrinodis, Myrmica schencki, Myrmica lobicornis and Myrmica hellenica. Only at one site (NE Poland) was a significant heterogeneity in parasitation rates among Myrmica species detected. M. lobicornis was the most often infested ant there, which may suggest local specialisation of the butterfly. Overall low parasitism rates may explain the vulnerability of P. arion in Central Europe but further studies are also necessary.     

Expression of G-protein coupled receptors in <Emphasis Type="Italic">Escherichia coli</Emphasis> for structural studies

To elaborate a high-performance system for expression of genes of G-protein coupled receptors (GPCR), methods of direct and hybrid expression of 17 GPCR genes in Escherichia coli and selection of strains and bacteria cultivation conditions were investigated. It was established that expression of most of the target GPCR fused with the N-terminal fragment of OmpF or Mistic using media for autoinduction provides high output (up to 50 mg/liter).