Thermodynamic analysis of ferrous ion binding to Escherichia coli ferritin EcFtnA

Iron oxidation in the bacterial ferritin EcFtnA from Escherichia coli shows marked differences from its homologue human H-chain ferritin (HuHF). While the amino acid residues that constitute the dinuclear center in these proteins are highly conserved, EcFtnA has a third iron-binding site (C site) in close proximity to the dinuclear center that is seemingly responsible for these differences. Here, we describe the first thermodynamic study of Fe2+ binding to EcFtnA and its variants to determine the location of the primary ferrous ion-binding sites on the protein and to better understand the role of the third C site in iron binding. Isothermal titration calorimetric analyses of the wild-type protein reveal the presence of two main classes of binding sites in the pH range of 6.5-7.5, ascribed to Fe2+ binding, first at the A and then the B sites. Site-directed mutagenesis of ligands in the A, B, or C sites affects the apparent Fe2+-binding stoichiometries at the unaltered sites. The data imply some degree of inter- and intrasubunit negative cooperative interaction between sites. Unlike HuHF where only the A site initially binds Fe2+, both A and B sites in EcFtnA bind Fe2+, implying a role for the C site in influencing the binding of Fe2+ at the B site of the di-iron center of EcFtnA. The ITC equations describing a binding model for three classes of independent binding sites are reported here for the first time.

A chimeric N-terminal Escherichia coli C-terminal Rhodobacter sphaeroides FliG rotor protein supports bidirectional E coli flagellar rotation and chemotaxis

Flagellate bacteria such as Escherichia coli and Salmonella enterica serovar Typhimurium typically express 5 to 12 flagellar filaments over their cell surface that rotate in clockwise (CW) and counterclockwise directions. These bacteria modulate their swimming direction towards favorable environments by biasing the direction of flagellar rotation in response to various stimuli. In contrast, Rhodobacter sphaeroides expresses a single subpolar flagellum that rotates only CW and responds tactically by a series of biased stops and starts. Rotor protein FliG transiently links the MotAB stators to the rotor, to power rotation and also has an essential function in flagellar export. In this study, we sought to determine whether the FliG protein confers directionality on flagellar motors by testing the functional properties of R. sphaeroides FliG and a chimeric FliG protein, EcRsFliG (N-terminal and central domains of E. coli FliG fused to an R. sphaeroides FliG C terminus), in an E. coli FliG null background. The EcRsFliG chimera supported flagellar synthesis and bidirectional rotation; bacteria swam and tumbled in a manner qualitatively similar to that of the wild type and showed chemotaxis to amino acids. Thus, the FliG C terminus alone does not confer the unidirectional stop-start character of the R. sphaeroides flagellar motor, and its conformation continues to support tactic, switch-protein interactions in a bidirectional motor, despite its evolutionary history in a bacterium with a unidirectional motor.

DNA interaction and phosphotransfer of the C-4-dicarboxylate-responsive DcuS-DcuR two-component regulatory system from Escherichia coli

The DcuS-DcuR system of Escherichia coli is a two-component sensor-regulator that controls gene expression in response to external C-4-dicarboxylates and citrate. The DcuS protein is particularly interesting since it contains two PAS domains, namely a periplasmic C-4-dicarboxylate-sensing PAS domain (PASp) and a cytosolic PAS domain (PASc) of uncertain function. For a study of the role of the PASc domain, three different fragments of DcuS were overproduced and examined: they were PASc-kinase, PASc, and kinase. The two kinase-domain-containing fragments were autophosphorylated by [gamma-P-32]ATP. The rate was not affected by fumarate or succinate, supporting the role of the PASp domain in C-4-dicarboxylate sensing. Both of the phosphorylated DcuS constructs were able to rapidly pass their phosphoryl groups to DcuR, and after phosphorylation, DcuR dephosphorylated rapidly. No prosthetic group or significant quantity of metal was found associated with either of the PASc-containing proteins. The DNA-binding specificity of DcuR was studied by use of the pure protein. It was found to be converted from a monomer to a dimer upon acetylphosphate treatment, and native polyacrylamide gel electrophoresis suggested that it can oligomerize. DcuR specifically bound to the promoters of the three known DcuSR-regulated genes (dctA, dcuB, and frdA), with apparent K(D)s of 6 to 32 muM for untreated DcuR and less than or equal to1 to 2 muM for the acetylphosphate-treated form. The binding sites were located by DNase I footprinting, allowing a putative DcuR-binding motif [tandemly repeated (T/A)(A/T)(T/C)(A/T)AA sequences] to be identified. The DcuR-binding sites of the dcuB, dctA, and frdA genes were located 27, 94, and 86 bp, respectively, upstream of the corresponding +1 sites, and a new promoter was identified for dcuB that responds to DcuR.

Characterization of an escherichia coli elaC deletion mutant

The elaC gene of Escherichia coli encodes a binuclear zinc phosphodiesterase (ZiPD). ZiPD homologs from various species act as 3' tRNA processing endoribonucleases, and although the homologous gene in Bacillus subtilis is essential for viability [EMBO J. 22 (2003) 4534], the physiological function of E. coli ZiPD has remained enigmatic. In order to investigate the function of E. coli ZiPD we generated and characterized an E. coli elaC deletion mutant. Surprisingly, the E. coli elaC deletion mutant was viable and had wild-type like growth properties. Micro array-based transcriptional analysis indicated expression of the E. coli elaC gene at basal levels during aerobic growth. The elaC gene deletion had no effect on the expression of genes coding for RNases or amino-acyl tRNA synthetases or any other gene among a total of > 1300 genes probed. 2D-PAGE analysis showed that the elaC mutation, likewise, had no effect on the proteome. These results strengthen doubts about the involvement of E. coli ZiPD in tRNA maturation and suggest functional diversity within the ZiPD/ElaCl protein family. In addition to these unexpected features of the E. coli elaC deletion mutant, a sequence comparison of ZiPD (ElaCl) proteins revealed specific regions for either enterobacterial or mammalian ZiPD (ElaCl) proteins. (C) 2004 Elsevier Inc. All rights reserved.

Mixed culture fermentation studies on the effects of synbiotics on the human intestinal pathogens Campylobacter jejuni and Escherichia coli

Batch and continuous culture anaerobic fermentation systems, inoculated with human faeces, were utilised to investigate the antimicrobial actions of two probiotics, Lactobacillus plantartan 0407, combined with oligofructose and Bifidobacterium bifidum Bb12, combined with a mixture of oligofructose and xylo-oligosaccharides (50:50 w/w) against E coli and Campylobacter jejuni. In batch fermenters, both E coli and C jejuni were inhibited by the synbiotics, even when the culture pH was maintained at around neutral. In continuous culture C jejuni was inhibited but the synbiotic failed to inhibit E coli. Although no definitive answer in addressing the mechanisms underlying antimicrobial activity was derived, results suggested that acetate and lactate directly were conferring antagonistic action, rather than as a result of lowering culture pH. In the course of the study culturing and fluorescent in situ hybridisation (FISH) methodologies for the enumeration of bacterial populations were compared. Bifidobacterial populations were underestimated using plating techniques, suggesting the non-culturability of certain bifidobacterial species. (C) 2003 Elsevier Ltd. All rights reserved.

Caseinoglycomacropeptide inhibits adhesion of pathogenic Escherichia coli strains to human cells in culture

Caseinoglycomacropeptide (CGMP) derived from kappa-casein was investigated for its ability to inhibit the adhesion of 3 strains of verotoxigenic Escherichia coli (VTEC) and 3 strains of enteropathogenic Escherichia coli (EPEC) to human HT29 tissue cell cultures. Effects on adhesion of Desulfovibrio desulfuricans, Lactobacillus pentosus, Lactobacillus casei, Lactobacillus acidophilus, and Lactobacillus gasseri were also investigated. Generally, CGMP exerted effective anti-adhesive properties at a dose of 2.5 mg/mL, albeit with a high degree of strain specificity. The CGMP reduced adhesion of VTEC strains to < 50% of the control and reduced adhesion of EPEC strains to between 80 and 10% of the control. The CGMP also reduced the adhesion of L. pentosus and L. casei to 44 and 42%, respectively. A slight but significant reduction of L. acidophilus, to 81%, was observed, but no significant effects were detected with either Dsv. desulfuricans or L. gasseri. Further investigation of the dose response relationships with the E. coli strains gave IC50 values ranging between 0.12 and 1.06 mg/mL.

Oligosaccharide-mediated inhibition of the adhesion of pathogenic Escherichia coli strains to human gut epithelial cells in vitro

The aim of the study was to investigate the ability of pectic oligosaccharides (POS) to inhibit adhesion of three strains of verotoxigenic Escherichia coli, three strains of enteropathogenic E. coli, and one nonclinical strain of Desulfovibrio desulfuricans to human intestinal epithelial cell cultures. Lactobacillus acidophilus and Lactobacillus gasseri were included for comparison. Attachment wits determined in the human HT29 cell line by viable Count of adherent bacteria. POS in buffer at pH 7.2 were antiadhesive at a dose of 2.5 mg ml(-1), reducing adhesion of enteropathogenic E. coli and verotoxigenic E. coli strains to less than 30% of control values. Concentrations resulting in 50% inhibition ranged from 0.15 to 0.46 mg ml(-1). L. acidophilus was not significantly affected. but adhesion of L. gasseri was reduced to 29% of the control value. POS reduced the adhesion of D. desulfuricans to 0.33% of the control value. POS also had a protective effect against E. coli verocytotoxins VT1 and VT2 at concentrations of 0.01 and 1 mu g ml(-1), respectively.

Inhibition of the adhesion of enteropathogenic Escherichia coli strains to HT-29 cells in culture by chito-oligosaccharides

The ability of chito-oligosaccharides (COS) to inhibit selected intestinal bacteria was investigated. COS at 2.5 mg ml(-1) had no significant effect on the adhesion of three strains of verotoxigenic Escherichia coli (VTEC), Lactobacillus pentosus, L. casei or L. gasseri to human HT29 cells in tissue culture. However, COS significantly inhibited adhesion of three strains of enteropathogenic E. coli (EPEC) to below 30% of the level of adhesion seen in the controls. Dose-response curves were constructed to further characterise the inhibition of EPEC strains to HT29 cells. (c) 2005 Elsevier Ltd. All rights reserved.

Pectins and pectic-oligosaccharides inhibit Escherichia coli O157 : H7 Shiga toxin as directed towards the human colonic cell line HT29

Pectins and pectic-oligosaccharides, as derived by controlled enzymatic hydrolysis, were evaluated for their ability to interfere with the toxicity of Shiga-like toxins from Escherichia coli O157:H7. Both types of material resulted in some degree of protection but this was significantly higher (P > 0.01) with the oligosaccharide fractions (giving 90-100% cell survival, compared to 70-80% with the polymer). An effect of methylation on the protective effect was detected with lower degrees being more active. The pectic-oligosaccharides and galabiose, the minimum toxin receptor analogue, were shown to inhibit toxicity and were both protective at 10 mg ml(-1), but not at lower concentrations. (C) 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Influence of environmental stress on distributions of times to first division in Escherichia coli populations, as determined by digital-image analysis of individual cells

The distributions of times to first cell division were determined for populations of Escherichia coli stationary-phase cells inoculated onto agar media. This was accomplished by using automated analysis of digital images of individual cells growing on agar and calculation of the "box area ratio." Using approximately 300 cells per experiment, the mean time to first division and standard deviation for cells grown in liquid medium at 37 degrees C and inoculated on agar and incubated at 20 degrees C were determined as 3.0 h and 0.7 h, respectively. Distributions were observed to tail toward the higher values, but no definitive model distribution was identified. Both preinoculation stress by heating cultures at 50 degrees C and postinoculation stress by growth in the presence of higher concentrations of NaCl increased mean times to first division. Both stresses also resulted in an increase in the spread of the distributions that was proportional to the mean division time, the coefficient of variation being constant at approximately 0.2 in all cases. The "relative division time," which is the time to first division for individual cells expressed in terms of the cell size doubling time, was used as measure of the "work to be done" to prepare for cell division. Relative division times were greater for heat-stressed cells than for those growing under osmotic stress.

Ribosome modulation factor protects Escherichia coli during heat stress, but this may not be dependent on ribosome dimerisation

The role of ribosome modulation factor (RMF) in protecting heat-stressed Escherichia coli cells was identified by the observation that cultures of a mutant strain lacking functional RMF (HMY15) were highly heat sensitive in stationary phase compared to those of the parent strain (W3110). No difference in heat sensitivity was observed between these strains in exponential phase, during which RMF is not synthesised. Studies by differential scanning calorimetry demonstrated that the ribosomes of stationary-phase cultures of the mutant strain had lower thermal stability than those of the parent strain in stationary phase, or exponential-phase ribosomes. More rapid breakdown of ribosomes in the mutant strain during heating was confirmed by rRNA analysis and sucrose density gradient centrifugation. Analyses of ribosome composition showed that the 100S dimers dissociated more rapidly during heating than 70S particles. While ribosome dimerisation is a consequence of the conformational changes caused by RMF binding, it may not therefore be essential for RMF-mediated ribosome stabilisation.

Morphological and physiological changes induced by high hydrostatic pressure in exponential- and stationary-phase cells of Escherichia coli: relationship with cell death

The relationship between a loss of viability and several morphological and physiological changes was examined with Escherichia coli strain J1 subjected to high-pressure treatment. The pressure resistance of stationary-phase cells was much higher than that of exponential-phase cells, but in both types of cell, aggregation of cytoplasmic proteins and condensation of the nucleoid occurred after treatment at 200 MPa for 8 min. Although gross changes were detected in these cellular structures, they were not related to cell death, at least for stationary-phase cells. In addition to these events, exponential-phase cells showed changes in their cell envelopes that were not seen for stationary-phase cells, namely physical perturbations of the cell envelope structure, a loss of osmotic responsiveness, and a loss of protein and RNA to the extracellular medium. Based on these observations, we propose that exponential-phase cells are inactivated under high pressure by irreversible damage to the cell membrane. In contrast, stationary-phase cells have a cytoplasmic membrane that is robust enough to withstand pressurization up to very intense treatments. The retention of an intact membrane appears to allow the stationary-phase cell to repair gross changes in other cellular structures and to remain viable at pressures that are lethal to exponential-phase cells.

Mixed culture fermentation studies on the effects of synbiotics on the human intestinal pathogens Campylobacter jejuni and Escherichia coli

Batch and continuous culture anaerobic fermentation systems, inoculated with human faeces, were utilised to investigate the antimicrobial actions of two probiotics, Lactobacillus plantartan 0407, combined with oligofructose and Bifidobacterium bifidum Bb12, combined with a mixture of oligofructose and xylo-oligosaccharides (50:50 w/w) against E coli and Campylobacter jejuni. In batch fermenters, both E coli and C jejuni were inhibited by the synbiotics, even when the culture pH was maintained at around neutral. In continuous culture C jejuni was inhibited but the synbiotic failed to inhibit E coli. Although no definitive answer in addressing the mechanisms underlying antimicrobial activity was derived, results suggested that acetate and lactate directly were conferring antagonistic action, rather than as a result of lowering culture pH. In the course of the study culturing and fluorescent in situ hybridisation (FISH) methodologies for the enumeration of bacterial populations were compared. Bifidobacterial populations were underestimated using plating techniques, suggesting the non-culturability of certain bifidobacterial species. (C) 2003 Elsevier Ltd. All rights reserved.