The mechanisms of activation of normal human serum complement by<Emphasis Type="Italic">Escherichia coli</Emphasis> strains with K1 surface antigen

Ten E. coli K1 strains isolated from the urine of children with urinary tract infections were sensitive to the bactericidal action of normal human serum (NHS). The role of the particular mechanisms of complement activation was determined in the process of killing these strains, showing variable sensitivity to the bactericidal action of NHS; three mechanisms of activation of human complement were observed. Important role of alternative pathway activation in the bactericidal action of NHS against E. coli K1 strains independent of the classical and lectin pathways was not established.     

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.     

High cell density fed-batch cultivation of<Emphasis Type=Italic> Escherichia coli</Emphasis> using exponential feeding combined with pH-stat

A new feeding strategy in fed-batch culture, exponential feeding combined with pH-stat is suggested to avoid the accumulation of substrate in culture broth. Exponential feeding was stopped whenever a predetermined amount of limiting substrate was supplied and then pH change was observed. When pH rose above an upper limit due to the depletion of substrate, feeding was restarted. With this feeding strategy, recombinant Escherichia coli could be grown to 101 g/l by controlling the specific growth rate at 0.1 h^–1.An erratum to this article can be found at     

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.     

Across Genus Plasmid Transformation Between <Emphasis Type=Italic>Bacillus subtilis</Emphasis> and <Emphasis Type=Italic>Escherichia coli</Emphasis> and the Effect of <Emphasis Type=Italic>Escherichia coli</Emphasis> on the Transforming Ability of Free Plasmid DNA

The results presented in this article show that direct plasmid transfer from Escherichia coli carrying shuttle plasmid to Bacillus subtilis occurred when close contact between the two species was established by mixing E. coli and B. subtilis onto selective agar plates. The data demonstrate that the production of resistant colonies by plasmid transformation through cell contact was DNase I sensitive and dependent on transformable B. subtilis strains. Furthermore, another observation indicated that the E. coli strain is able to affect the transformation capability of B. subtilis. It is assumed that the donor strain is a momentous factor for taking up plasmid DNA. This conclusion is significant in the assessment of both the possibility of intercellular DNA transfer in natural habitats of micro-organisms and the risk of the application of genetically engineered micro-organisms.     

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.     

Physiology of<Emphasis Type=Italic>Anabaena khannae</Emphasis> and<Emphasis Type=Italic>Chlorococcum humicola</Emphasis> under fluoride stress

Sodium fluoride showed pH-dependent physiological responses in the two test microalgae Anabaena khannae and Chlorococcum humicola. A. khannae showed severe membrane damage with fluoride at low pH with leakage of pigments and electrolytes. Annihilation of photosynthesis along with inhibition in 14C uptake was observed at pH 6 with 50 mg/L fluoride. While respiration was less affected in the cyanobacterium, C. humicola showed 30 % inhibition in respiratory activity. Resistance of C. humicola to fluoride toxicity has been attributed to the hindrance provided by the thick cell envelope, intracellular compartmentation and increase in extracellular pH as a consequence of its metabolism.     

Localization of replication forks in wild-type and <Emphasis Type=Italic>mukB</Emphasis> mutant cells of <Emphasis Type=Italic>Escherichia coli</Emphasis>

To examine the subcellular localization of the replication machinery in Escherichia coli, we have developed an immunofluorescence method that allows us to determine the subcellular location of newly synthesized DNA pulse-labeled with 5-bromo-2′-deoxyuridine (BrdU). Using this technique, we have analyzed growing cells. In wild-type cells that showed a single BrdU fluorescence signal, the focus was located in the middle of the cell; in cells with two signals, the foci were localized at positions equivalent to 1/4 and 3/4 of the cell length. The formation of BrdU foci was dependent upon ongoing chromosomal replication. A mutant lacking MukB, which is required for proper partitioning of sister chromosomes, failed to maintain the ordered localization of BrdU foci: (1) a single BrdU focus tended to be localized at a pole-proximal region of the nucleoid, and (2) a focus was often found to consist of two replicating chromosomes. Thus, the positioning of replication forks is affected by the disruption of the mukB gene.     

The diagnostic implications of the separation of<Emphasis Type=Italic>Entamoeba histolytica</Emphasis> and<Emphasis Type=Italic>Entamoeba dispar</Emphasis>

For much of the last hundred years most cases of amoebiasis have been diagnosed by light microscopy. Only relatively recently have we become aware that this technique is usually incapable of distinguishing between two species-Entamoeba histolytica andE. dispar-only the first of which is a pathogen. The implications of this for patient management and, even more, for the validity of epidemiological surveys, are only slowly being addressed. What is clear is that methods are urgently required to distinguish between infections with these two species and this review attempts to summarise some of those, which have been developed to meet this need.     

Expression of PHA polymerase genes of <Emphasis Type=Italic>Pseudomonas putida</Emphasis> in <Emphasis Type=Italic>Escherichia coli</Emphasis> and its effect on PHA formation

Poly-3-hydroxyalkanoates (PHAs) are synthesized by many bacteria as intracellular storage material. The final step in PHA biosynthesis is catalyzed by two PHA polymerases (phaC) in Pseudomonas putida. The expression of these two phaC genes (phaC1 and phaC2)was studied in Escherichia coli, either under control of the native promoter or under control of an external promoter. It was found that the two phaC genes are not expressed in E. coli without an external promoter. During heterologous expression of phaC from Plac on a high copy number plasmid, a rapid reduction of the number of colony forming units was observed, especially for phaC2. It appears that the plasmid instability was partially caused by high-level production of PHA polymerase. Subsequently, tightly regulated phaC2 expression systems on a low copy number vector were applied in E. coli. This resulted in PHA yields of over 20 of total cell dry weight, which was 2 fold higher than that obtained from the system where phaC2 is present on a high copy number vector. In addition, the PHA monomer composition differed when different gene expression systems or different phaC genes were applied.     

Production of Vanillin by Metabolically Engineered <Emphasis Type=Italic>Escherichia coli</Emphasis>

E. coli was metabolically engineered to produce vanillin by expression of the fcs and ech genes from Amycolatopsis sp. encoding feruloyl-CoA synthetase and enoyl-CoA hydratase/aldolase, respectively. Vanillin production was optimized by leaky expression of the genes, under the IPTG-inducible trc promoter, in complex 2YT medium. Supplementation with glucose, fructose, galactose, arabinose or glycerol severely decreased vanillin production. The highest vanillin production of 1.1 g l⁻¹ was obtained with cultivation for 48 h in 2YT medium with 0.2% (w/v) ferulate, without IPTG and no supplementation of carbon sources.     

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.     

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.     

Involvement of <Emphasis Type=Italic>soxRS</Emphasis> Regulon in Response of <Emphasis Type=Italic>Escherichia coli</Emphasis> to Oxidative Stress Induced by Hydrogen Peroxide

The effect of hydrogen peroxide on the activity of soxRS and oxyR regulon enzymes in different strains of Escherichia coli has been studied. Treatment of bacteria with 20 μM H₂O₂ caused an increase in catalase and peroxidase activities (oxyR regulon) in all strains investigated. It is shown for the first time that oxidative stress induced by hydrogen peroxide causes in some E. coli strains a small increase in activity of superoxide dismutase and glucose-6-phosphate dehydrogenase (soxRS regulon). This effect is cancelled by chloramphenicol, an inhibitor of protein synthesis in prokaryotes. The increase in soxRS regulon enzyme activities was not found in the strain lacking the soxR gene. These results provide evidence for the involvement of the soxRS regulon in the adaptive response of E. coli to oxidative stress induced by hydrogen peroxide. __________ Translated from Biokhimiya, Vol. 70, No. 11, 2005, pp. 1506–1513. Original Russian Text Copyright ? 2005 by Semchyshyn, Bagnyukova, Lushchak.     

Secretory production of<Emphasis Type=Italic> Aspergillus oryzae</Emphasis> xylanase XynF1,<Emphasis Type=Italic> xynF1</Emphasis> cDNA product,in the basidiomycete<Emphasis Type=Italic> Coprinus cinereus</Emphasis>

The signal peptide of Aspergillus oryzae endo-(1,4)--xylanase XynF1 contains a C-terminal serine-arginine that directs efficient secretion of the enzyme into the culture medium. In the basidiomycete Coprinus cinereus, however, there is little secretion of XynF1 into the culture medium. Modification of the C-terminal sequence of the signal peptide to lysine-arginine resulted in efficient secretion of C. cinereus XynF1, suggesting the presence of a KEX2-like protease in this fungus.     

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.     

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.