The KefB and KefC systems of E. coli are two separate potassium efflux systems that are regulated by glutathione and specific glutathione metabolites. The aims of this study were to clone and sequence the E. coli kefB structural gene and to investigate the subunit stoichiometry of the E. coli KefC protein. A further objective was to determine the distribution of the KefC class of transport system in a range of bacterial species, with the ultimate aim of cloning the kefC homologue from a highly divergent organism. Attempts to clone the kefB gene by various strategies proved unsuccessful; possible explanations for this are discussed. Analysis of the suppression of a kefC leaky mutation by plasmids bearing varying lengths of the wild-type kefC gene indicated that the mutant and wild-type KefC proteins interact in the membrane to form a structure that has intermediate properties. These observations are consistent with KefC functioning as an oligomer. Using a variety of techniques, it was demonstrated that the KefC class of transport system is widely distributed among Gram-negativespecies but is absent from all the Gram-positive bacteria tested with the exception of Staph. aureus. In addition the kefC homologue from Er. carotovora was cloned and DNA sequencing initiated. The cloned Er. carotovora kefC gene product was able to suppress an E. coli kefC leaky mutation, thus providing further evidence that KefC exists as an oligomer.

Wild type cells of <i>Escherichia coli</i> K-12 are unable to grow on glutamate as the sole source of carbon. Glutamate-utilising mutants have been isolated previously and found to exhibit enhanced glutamate transport. Mutations at two loci <i>gltS</i> and <i>gltR</i> and possibly a third <i>gltC</i> were previously shown by other workers to mediate enhanced glutamate permeability. The <i>gltR</i> locus was suggested to be a negative regulatory for the <i>gltS</i> gene. A mutation at the <i>gltS</i> locus, the <i>gltSo</i> mutation increases the activity of a Na⁺-stimulated glutamate transport system GltI. The regulation of the GltI system was investigated by the isolation of MudX gene fusions which abolished Na⁺-stimulated glutamate transport. Two fusions with 'strong' and 'weak' B-galactosidase activities were isolated. These were both found to map at the <i>gltS</i> locus. Subsequent mapping exercises, suggested that the MudX fusions may be located in separate genes. Transcription of the 'strong' fusion was found to be impaired in the presence of the <i>phs</i> mutation. This is a mutation in the <i>rpoA</i> gene which encodes the -subunit of RNA polymerase. Glutamate-utilising suppressor mutants were isolated in both the MudX gene fusion strains. The suppressor mutations were found to map at the <i>gltR</i> locus. This was found to be the map location of a second glutamate transport system GltII. Thus, the <i>gltR</i> locus was found not to be the location of a negative regulator for the <i>gltS</i> gene. The <i>melAB</i> operon encodes the proteins for melibiose transport and utilisation. No regulatory locus has previously been reported for this transport system. The regulation of the <i>melAB</i> operon was investigated by cloning the <i>melAB</i> promoter into an <i>lacZ</i> expression vector. The region upstream from the <i>melAB</i> promoter was subsequently found to encode a trans-acting positive regulatory necessary for <i>melAB</i> expression. Transcription from the <i>melAB</i> promoter was also found to be impaired by the <i>phs</i> mutation. The results presented in this study substantiate previous observations that the <i>phs</i> mutation causes a generalised transcription defect.

Enhanced transport of microbes in subsurface is a focus in bioaugmentation applications for remediation of groundwater. In this study, the effect of low-concentration monorhamnolipid biosurfactant on transport of Pseudomonas aeruginosa ATCC 9027 in natural porous media (silica sand and a sandy soil) with or without hexadecane as the nonaqueous phase liquids (NAPLs) was studied with miscible-displacement experiments using artificial groundwater as the background solution. Transport of two types of cells was investigated, glucose-grown and hexadecane-grown cells with lower and higher cell surface hydrophobicity (CSH), respectively. A clean-bed colloid deposition model was used to calculate deposition rate coefficients (k) for quantitative assessment on the effect of the rhamnolipid on the transport. In the absence of NAPLs, significant cell retention was observed in the sand (81% and 82% for glucose-grown and hexadecane-grown cells, respectively). Addition of low-concentration rhamnolipid enhanced cell transport, with 40 mg/L of rhamnolipid reducing retention to 50% and 60% for glucose-grown and hexadecane-grown cells, respectively. The k values for both glucose-grown and hexadecane-grown cells correlated linearly with rhamnolipid-dependent CSH quantitatively measured using a bacterial-adhesion-to-hydrocarbon method. Retention of cells by the soil was nearly complete (>99%). Forty milligrams per liter of rhamnolipid reduced the retention to 95%. The presence of NAPLs in the sand enhanced the retention of hexadecane-grown cells with higher CSH. Transport of cells in the presence of NAPLs was enhanced by rhamnolipid at all concentrations tested, and the relative enhancement was greater than in the absence of NAPLs. This study shows the importance of hydrophobic interaction on bacterial transport in natural porous media and the potential of using low-concentration rhamnolipid for facilitating cell transport in subsurface for bioaugmentation efforts.

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No description available.

Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine, with therapeutic applications in inflammatory bowel disease. For the in situ delivery of IL-10 by Escherichia coli as carrier chassis, a modified transporter was designed with the ability to secrete biologically active IL-10. De novo DNA synthesis comprised a 561-bp fragment encoding the signal sequence of the E. coli outer membrane protein F fused in frame to an E. coli codon-optimized mature human IL-10 gene under control of a T7 promoter. The construct was overexpressed in E. coli laboratory strains, E. coli BL21 (DE3) and E. coli MDS42:T7. The mean concentrations of human IL-10 in the periplasm and culture supernatant of E. coli BL21 (DE3) were 355.8 ± 86.3 and 5.7 ± 1.7 ng/ml, respectively. The molecular mass of the recombinant E. coli-derived human IL-10 was 19 kDa, while under non-reducing conditions the native IL-10 dimer could be demonstrated. Reduction of tumor necrosis factor-α secretion in lipopolysaccharide-stimulated mouse macrophages and detection of the activated form of the transcription factor signal transducer and activator of transcription protein 3 proved the biological activity of the bacteria-produced human IL-10. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Models of the fate and transport of river plumes and the bacteria they carry into lakes are developed. They are needed to enable informed decisions about beach closures to avoid economic losses, and to help design water intakes and operate combined sewer overflow schemes to obviate exposure of the public to potential pathogens. This study advances our understanding of river plumes dynamics in coastal waters by means of field studies and numerical techniques. Extensive field measurements were carried out in the swimming seasons of 2006 and 2007 on the Grand River plume as it enters Lake Michigan. They included simultaneous aerial photography, measurements of lake physical properties, the addition of artificial tracers to track the plume, and bacterial sampling. Our observed results show more flow classes than included in previous studies (e.g. CORMIX). Onshore wind can have a significant effect on the plume and whether it impacts the shoreline. A new classification scheme based on the relative magnitude of plume-crossflow length scale and Richardson number based on the wind speed is devised. Previous studies on lateral spreading are complemented with a new relationship in the near field. The plume thickness decreased rapidly with distance from the river mouth and a new non-dimensional relationship to predict thickness is developed. Empirical near field models for surface buoyant plumes are reviewed and a near field trajectory and dilution model for large aspect ratio surface discharge channels is devised. Bacterial reductions due to dilution were generally small (less than 10:1) up to 4.5 km from the river mouth. E. coli decay rates were significantly affected by solar radiation and ranged from 0.2 to 2.2 day-1 which were within the range of previous studies in Lake Michigan. Total coliform survived longer than E. coli suggesting different die-off mechanisms. Mathematical models of the bacterial transport are developed that employ a nested modeling scheme to represent the 3D hydrodynamic processes of surface river discharges in the Great Lakes. A particle tracking model is used that provides the capability to track a decaying tracer and better quantify mixing due to turbulent diffusion. Particle tracking models have considerable advantages over gradient diffusion models in simulating bacterial behavior nearshore that results in an improved representation of bacteria diffusion, decay and transport. Due to the complexity and wide variation of the time and length scale of the hydrodynamic and turbulent processes in the near field (where plume mixing is dominated by initial momentum and buoyancy) and far field (where plume mixing is dominated by ambient turbulence), a coupling technique is adapted. The far field random walk particle tracking model incorporates the empirical near field model. It simulates the transport, diffusion and decay of bacteria as discrete particles and employs the near field output as the source and transports the particles based on ambient currents predicted by the 3D hydrodynamic model. The coupled model improves dilution predictions in the near field. The new techniques advance our knowledge of the nearshore fate and transport of bacteria in the Great Lakes and can be ultimately applied to the NOAA Great Lakes Coastal Forecasting System to provide a reliable prediction tool for bacterial transport in recreational waters.

Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine, with therapeutic applications in inflammatory bowel disease. For the in situ delivery of IL-10 by Escherichia coli as carrier chassis, a modified transporter was designed with the ability to secrete biologically active IL-10. De novo DNA synthesis comprised a 561-bp fragment encoding the signal sequence of the E. coli outer membrane protein F fused in frame to an E. coli codon-optimized mature human IL-10 gene under control of a T7 promoter. The construct was overexpressed in E. coli laboratory strains, E. coli BL21 (DE3) and E. coli MDS42:T7. The mean concentrations of human IL-10 in the periplasm and culture supernatant of E. coli BL21 (DE3) were 355.8 ± 86.3 and 5.7 ± 1.7 ng/ml, respectively. The molecular mass of the recombinant E. coli-derived human IL-10 was 19 kDa, while under non-reducing conditions the native IL-10 dimer could be demonstrated. Reduction of tumor necrosis factor-α secretion in lipopolysaccharide-stimulated mouse macrophages and detection of the activated form of the transcription factor signal transducer and activator of transcription protein 3 proved the biological activity of the bacteria-produced human IL-10. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.