Cross-linking studies with the uvrA and uvrB proteins of E. coli

The interactions of the uvrA and uvrB proteins with DNA have been investigated using a DNA-protein cross-linking technique. It is demonstrated that hydrolysis of ATP by the uvrA protein facilitates cross-linking of this protein to single-stranded DNA, whether the DNA is UV irradiated or not. In contrast, cross-linking to unirradiated double-stranded DNA is not facilitated by ATP hydrolysis and is in fact increased by the substitution of the non-hydrolysable analogue aTP gamma S for ATP. In the presence of ATP, a dose-dependent increase is observed in the amount of uvrA protein which can be cross-linked to UV-irradiated double-stranded DNA. Binding of uvrB protein to puvrA-DNA complexes has a stabilising effect and increases the number of complexes which can be cross-linked whether the substrate is single- or double-stranded DNA. We can find no evidence that ATP hydrolysis by uvrA protein results in unwinding of UV-damaged DNA.     

Variation of the folding and dynamics of the Escherichia coli chromosome with growth conditions

We examine whether the Escherichia coli chromosome is folded into a self‐adherent nucleoprotein complex, or alternately is a confined but otherwise unconstrained self‐avoiding polymer. We address this through in vivo visualization, using an inducible GFP fusion to the nucleoid‐associated protein Fis to non‐specifically decorate the entire chromosome. For a range of different growth conditions, the chromosome is a compact structure that does not fill the volume of the cell, and which moves from the new pole to the cell centre. During rapid growth, chromosome segregation occurs well before cell division, with daughter chromosomes coupled by a thin inter‐daughter filament before complete segregation, whereas during slow growth chromosomes stay adjacent until cell division occurs. Image correlation analysis indicates that sub‐nucleoid structure is stable on a 1 min timescale, comparable to the timescale for redistribution time measured for GFP–Fis after photobleaching. Optical deconvolution and writhe calculation analysis indicate that the nucleoid has a large‐scale coiled organization rather than being an amorphous mass. Our observations are consistent with the chromosome having a self‐adherent filament organization.     

Escherichia coli DnaA interacts with HU in initiation at the E. coli replication origin

Escherichia coli HU protein is a dimer encoded by two closely related genes whose expression is growth phase-dependent. As a major component of the bacterial nucleoid, HU binds to DNA non-specifically, but acts at the chromosomal origin (oriC) during initiation by stimulating strand opening in vitro. We show that the alpha dimer of HU is more active than other forms of HU in initiation of an oriC-containing plasmid because it more effectively promotes strand opening of oriC. Other results demonstrate that HU stabilizes the DnaA oligomer bound to oriC, and that the alpha subunit of HU interacts with the N-terminal region of DnaA. These observations support a model whereby DnaA interacts with the alpha dimer or the alphabeta heterodimer, depending on their cellular abundance, to recruit the respective form of HU to oriC. The greater activity of the alpha dimer of HU at oriC may stimulate initiation during early log phase compared with the lesser activity of the alphabeta heterodimer or the beta dimer.     

Isolation and characterization of the outer membrane of Escherichia coli with autodisplayed Z-domains

“Autodisplay technology” is an expression technique used to display the various recombinant proteins on the outer membrane (OM) of Escherichia coli. The resulting autodisplayed Z-domain has been used to improve the sensitivity of immunoassays. In this work, a facile isolation method of the OM fraction of E. coli with autodisplayed Z-domains was presented using (1) an enzyme reaction for the hydrolysis of the peptidoglycan layer and (2) short centrifugation steps. The purity of the isolated OM fraction was analyzed. For the estimation of contamination with bacterial proteins from other parts of E. coli, Western blots of marker proteins for the OM (OmpA), periplasm (β-lactamase), inner membrane (SecA), and cytoplasm (β-galactosidase) were performed. Additionally, assays of marker components or enzymes from each part of E. coli were carried out including the OM (KDO), inner membrane (NADH oxidase), periplasm (β-lactamase), and cytoplasm (β-galactosidase). The yield of OM isolation using this new method was determined to be 80% of the total OM amount, with less than 1% being contaminants from other parts of E. coli.     

Association of Enterohemorrhagic Escherichia coli Hemolysin with Serotypes of Shiga-Like-Toxin-Producing Escherichia coli of Human and Bovine Origins

In this study we investigated whether the enterohemorrhagic Escherichia coli (EHEC) hemolysin gene ehxA could be used as an indicator of pathogenicity in Shiga-like-toxin-producing Escherichia coli (SLTEC) isolates. The isolates in a collection of 770 SLTEC strains of human and bovine origins were assigned to group 1 (230 human and 138 bovine SLTEC isolates belonging to serotypes frequently implicated in human disease), group 2 (85 human and 183 bovine isolates belonging to serotypes less frequently implicated in disease), and group 3 (134 bovine isolates belonging to serotypes not implicated in disease). PCR amplification was used to examine all of the SLTEC isolates for the presence of ehxA and the virulence-associated genes eae, slt-I, and slt-II. The percentages of human isolates in groups 1 and 2 that were positive for ehxA were 89 and 46%, respectively, and the percentages of bovine isolates in groups 1 to 3 that were positive for ehxA were 89, 51, and 52%, respectively. The percentages of human isolates in groups 1 and 2 that were positive for eae were 92 and 27%, respectively, and the percentages of bovine isolates in groups 1 to 3 that were positive for eae were 78, 15, and 19%, respectively. The frequencies of both ehxA and eae were significantly higher for group 1 isolates than for group 2 isolates. The presence of the ehxA gene was associated with serotype, as was the presence of the eae gene. Some serotypes, such as O117:H4, lacked both eae and ehxA and have been associated with severe disease, but only infrequently. The slt-I genes were more frequent in group 1 isolates than in group 2 isolates, and the slt-II genes were more frequent in group 2 isolates than in group 1 isolates. In a second experiment we determined the occurrence of the ehxA and slt genes in E. coli isolated from bovine feces. Fecal samples from 175 animals were streaked onto washed sheep erythrocyte agar plates. Eight E. coli-like colonies representing all of the morphological types were transferred to MacConkey agar. A total of 1,080 E. coli isolates were examined, and the ehxA gene was detected in 12 independent strains, only 3 of which were positive for slt. We concluded that the ehxA gene was less correlated with virulence than the eae gene was and that EHEC hemolysin alone has limited value for screening bovine feces for pathogenic SLTEC because of presence of the ehxA gene in bovine isolates that are not SLTEC.     

Biocatalysis with immobilized Escherichia coli

Immobilization is one of the great tools for developing economically and ecologically available biocatalysts and can be applied for both enzymes and whole cells. Much research dealing with the immobilization of Escherichia coli has been published in the past two decades. E. coli in the form of immobilized biocatalyst catalyzes many interesting reactions and has been used mainly in laboratories, but also on an industrial scale, leading to the production of valuable substances. It has the potential to be applied in many fields of modern biotechnology. This paper aims to give a general overview of immobilization techniques and matrices suitable mostly for entrapment, encapsulation, and adsorption, which have been most frequently used for the immobilization of E. coli. An extensive analysis reviewing the history and current state of immobilized E. coli catalyzing different types of biotransformations is provided. The review is organized according to the enzymes expressed in immobilized E. coli, which were grouped into main enzyme classes. The industrial applications of immobilized E. coli biocatalyst are also discussed.     

Interactions of the Escherichia coli methionine repressor with the metF operator and with its corepressor, S-adenosylmethionine

The metJ gene encoding the methionine aporepressor was placed under the control of a strong and inducible promoter, ptac. Bacterial strains carrying the recombinant plasmid pIP35 overproduced the regulatory protein by a factor of 200 over the wild type strain as determined by the immunoblot technique. The purified metJ gene product negatively controls the expression of the metF gene, in a cell-free system as shown by repression of beta-galactosidase synthesis under the control of the metF promoter. The metJ protein binds to a DNA fragment containing the potential operator of the metF gene with an affinity which is 10 times greater in the presence of S-adenosylmethionine than in its absence. Equilibrium dialysis experiments showed that the met aporepressor binds 2 mol of S-adenosylmethionine per mol of dimer with a dissociation constant of 200 microM.     

CRISPR Content Correlates with the Pathogenic Potential of Escherichia coli

Guide RNA molecules (crRNA) produced from clustered regularly interspaced short palindromic repeat (CRISPR) arrays, altogether with effector proteins (Cas) encoded by cognate cas (CRISPR associated) genes, mount an interference mechanism (CRISPR-Cas) that limits acquisition of foreign DNA in Bacteria and Archaea. The specificity of this action is provided by the repeat intervening spacer carried in the crRNA, which upon hybridization with complementary sequences enables their degradation by a Cas endonuclease. Moreover, CRISPR arrays are dynamic landscapes that may gain new spacers from infecting elements or lose them for example during genome replication. Thus, the spacer content of a strain determines the diversity of sequences that can be targeted by the corresponding CRISPR-Cas system reflecting its functionality. Most Escherichia coli strains possess either type I-E or I-F CRISPR-Cas systems. To evaluate their impact on the pathogenicity of the species, we inferred the pathotype and pathogenic potential of 126 strains of this and other closely related species and analyzed their repeat content. Our results revealed a negative correlation between the number of I-E CRISPR units in this system and the presence of pathogenicity traits: the median number of repeats was 2.5-fold higher for commensal isolates (with 29.5 units, range 0–53) than for pathogenic ones (12.0, range 0–42). Moreover, the higher the number of virulence factors within a strain, the lower the repeat content. Additionally, pathogenic strains of distinct ecological niches (i.e., intestinal or extraintestinal) differ in repeat counts. Altogether, these findings support an evolutionary connection between CRISPR and pathogenicity in E. coli.     

Interactions of elongation factor EF-P with the Escherichia coli ribosome

EF-P (eubacterial elongation factor P) is a highly conserved protein essential for protein synthesis. We report that EF-P protects 16S rRNA near the G526 streptomycin and the S12 and mRNA binding sites (30S T-site). EF-P also protects domain V of the 23S rRNA proximal to the A-site (50S T-site) and more strongly the A-site of 70S ribosomes. We suggest that EF-P: (a) may play a role in translational fidelity and (b) prevents entry of fMet-tRNA into the A-site enabling it to bind to the 50S P-site. We also report that EF-P promotes a ribosome-dependent accommodation of fMet-tRNA into the 70S P-site.     

Treatment of inflammatory bowel disease associated E. coli with ciprofloxacin and E. coli Nissle in the streptomycin-treated mouse intestine

Background

E. coli belonging to the phylogenetic group B2 are linked to Inflammatory Bowel Disease (IBD). Studies have shown that antimicrobials have some effect in the treatment of IBD, and it has been demonstrated that E. coli Nissle has prophylactic abilities comparable to 5-aminosalicylic acid (5-ASA) therapy in ulcerative colitis. The objective of this study was to test if ciprofloxacin and/or E. coli Nissle could eradicate IBD associated E. coli in the streptomycin-treated mouse intestine.

Results

After successful colonization with the IBD associated E. coli strains in mice the introduction of E. coli Nissle did not result in eradication of either IBD associated strains or an E. coli from a healthy control, instead, co-colonization at high levels were obtained. Treatment of mice, precolonized with IBD associated E. coli, with ciprofloxacin for three days alone apparently resulted in effective eradication of tested E. coli. However, treatment of precolonized mice with a combination of ciprofloxacin for 3 days followed by E. coli Nissle surprisingly allowed one IBD associated E. coli to re-colonize the mouse intestine, but at a level 3 logs under E. coli Nissle. A prolonged treatment with ciprofloxacin for 7 days did not change this outcome.

Conclusions

In the mouse model E. coli Nissle can not be used alone to eradicate IBD associated E. coli; rather, 3 days of ciprofloxacin are apparently efficient in eradicating these strains, but surprisingly, after ciprofloxacin treatment (3 or 7 days), the introduction of E. coli Nissle may support re-colonization with IBD associated E. coli.     

Expression of the Escherichia coli K5 capsular antigen: immunoelectron microscopic and biochemical studies with recombinant E. coli.

The capsular K5 polysaccharide, a representative of group II capsular antigens of Escherichia coli, has been cloned previously, and three gene regions responsible for polymerization and surface expression have been defined (I. S. Roberts, R. Mountford, R. Hodge, K. B. Jann, and G. J. Boulnois, J. Bacteriol. 170:1305-1310, 1988). In this report, we describe the immunoelectron microscopic analysis of recombinant bacteria expressing the K5 antigen and of mutants defective in either region 1 or region 3 gene functions, as well as the biochemical analysis of the K5 capsular polysaccharide. Whereas the K5 clone expressed the K5 polysaccharide as a well-developed capsule in about 25% of its population, no capsule was observed in whole mount preparations and ultrathin sections of the expression mutants. Immunogold labeling of sections from the region 3 mutant revealed the capsular K5 polysaccharide in the cytoplasm. With the region 1 mutant, the capsular polysaccharide appeared associated with the cell membrane, and, unlike the region 3 mutant polysaccharide, the capsular polysaccharide could be detected in the periplasm after plasmolysis of the bacteria. Polysaccharides were isolated from the homogenized mutants with cetyltrimethylammonium bromide. The polysaccharide from the region 1 mutant had the same size as that isolated from the capsule of the original K5 clone, and both polysaccharides were substituted with phosphatidic acid. The polysaccharide from the region 3 mutant was smaller and was not substituted with phosphatidic acid. These results prompt us to postulate that gene region 3 products are involved in the translocation of the capsular polysaccharide across the cytoplasmic membrane and that region 1 directs the transport of the lipid-substituted capsular polysaccharide through the periplasm and across the outer membrane.     

Adenomatous polyposis coli associates with the microtubule-destabilizing protein XMCAK

During cell division, the proper formation of a bipolar spindle and its function to segregate chromosomes requires precise coordination of microtubule-stabilizing and destabilizing activities. Globally destabilized, dynamic microtubules radiating from duplicated centrosomes are locally regulated by chromosomes. Proteins at the kinetochore of each sister chromatid mediate a dynamic attachment, allowing chromosome movement coupled to microtubule polymerization/depolymerization and error-correction mechanisms for improperly attached chromosomes. The tumor suppressor protein adenomatous polyposis coli (APC) stabilizes microtubules both in vitro and in vivo and is implicated in mitosis, although its mechanisms of action are not well characterized. Here, we show that in mitotic Xenopus egg extracts, the carboxyl-terminus of APC can associate with the amino terminus of the microtubule-destabilizing KinI, Xenopus mitotic centromere-associated kinesin (XMCAK), in a cytoplasmic complex. We find that like XMCAK, APC can localize to the centromere as well as the kinetochore region of mitotic chromosomes and does not require microtubules for chromosomal targeting in Xenopus egg extracts. We propose that the presence of these proteins in a complex brings together both positive and negative microtubule effectors, whose opposing activities may be regulated by additional factors, thereby providing precise control of both global and local microtubule dynamics.     

ClpB proteins copurify with the anaerobic Escherichia coli reductase

Two proteins, called alpha and beta 3, copurify with the anaerobic ribonucleotide reductase from Escherichia coli (Eliasson et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3314-3318). Both are now identified as products of the clpB gene that is presumed to code for a subunit of an ATP dependent protease. The tight associations suggest the possibility that the ClpB proteins are involved in the regulation of the anaerobic reductase.