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Pharmacodynamics of Ceftazidime plus the Serine ß-Lactamase Inhibitor AM-112 against Escherichia coli Containing TEM-1 and CTX-M-1 ß-Lactamases. Karen E. Bowker, 2004.A strain of Escherichia coli containing TEM-1 and CTX-M-1 was tested in an in vitro pharmacokinetic model against ceftazidime with and without AM-112, a serine ß-lactamase inhibitor . Ceftazidime alone was less effective than ceftazidime plus AM-112, and a single dose was more effective than three fractionated doses . Model System for Growing and Quantifying Streptococcus pneumoniae Biofilms In Situ and in Real Time. R. M. Donlan, 2004.Streptococcus pneumoniae forms biofilms, but little is known about its extracellular polymeric substances (EPS) or the kinetics of biofilm formation . A system was developed to enable the simultaneous measurement of cells and the EPS of biofilm-associated S . pneumoniae in situ over time . A biofilm reactor containing germanium coupons was interfaced to an attenuated total reflectance (ATR) germanium cell of a Fourier transform infrared (FTIR) laser spectrometer . Biofilm-associated cells were recovered from the coupons and quantified by total and viable cell count methods . ATR-FTIR spectroscopy of biofilms formed on the germanium internal reflection element (IRE) of the ATR cell provided a continuous spectrum of biofilm protein and polysaccharide (a measure of the EPS) . Staining of the biofilms on the IRE surface with specific fluorescent probes provided confirmatory evidence for the biofilm structure and the presence of biofilm polysaccharides . Biofilm protein and polysaccharides were detected within hours after inoculation and continued to increase for the next 141 h . The polysaccharide band increased at a substantially higher rate than did the protein band, demonstrating increasing coverage of the IRE surface with biofilm polysaccharides . The biofilm total cell counts on germanium coupons stabilized after 21 h, at approximately 105 cells per cm2, while viable counts decreased as the biofilm aged . This system is unique in its ability to detect and quantify biofilm-associated cells and EPS of S . pneumoniae over time by using multiple, corroborative techniques . This approach could prove useful for the study of biofilm processes of this or other microorganisms of clinical or industrial relevance . Proteolytic Cleavage of the FlhB Homologue YscU of Yersinia pseudotuberculosis Is Essential for Bacterial Survival but Not for Type III Secretion. Moa Lavander, 2002.Pathogenic Yersinia species employ a type III secretion system (TTSS) to target antihost factors, Yop proteins, into eukaryotic cells . The secretion machinery is constituted of ca . 20 Ysc proteins, nine of which show significant homology to components of the flagellar TTSS . A key event in flagellar assembly is the switch from secreting-assembling hook substrates to filament substrates, a switch regulated by FlhB and FliK . The focus of this study is the FlhB homologue YscU, a bacterial inner membrane protein with a large cytoplasmic C-terminal domain . Our results demonstrate that low levels of YscU were required for functional Yop secretion, whereas higher levels of YscU lowered both Yop secretion and expression . Like FlhB, YscU was cleaved into a 30-kDa N-terminal and a 10-kDa C-terminal part . Expression of the latter in a wild-type strain resulted in elevated Yop secretion . The site of cleavage was at a proline residue, within the strictly conserved amino acid sequence NPTH . A YscU protein with an in-frame deletion of NPTH was cleaved at a different position and was nonfunctional with respect to Yop secretion . Variants of YscU with single substitutions in the conserved NPTH sequence—i.e., N263A, P264A, or T265A—were not cleaved but retained function in Yop secretion . Elevated expression of these YscU variants did, however, result in severe growth inhibition . From this we conclude that YscU cleavage is not a prerequisite for Yop secretion but is rather required to maintain a nontoxic fold . Residues C123 and D58 of the 2-Methylisocitrate Lyase (PrpB) Enzyme of Salmonella enterica Are Essential for Catalysis. T. L. Grimek, 2003.The prpB gene of Salmonella enterica serovar Typhimurium LT2 encodes a protein with 2-methylisocitrate (2-MIC) lyase activity, which cleaves 2-MIC into pyruvate and succinate during the conversion of propionate to pyruvate via the 2-methylcitric acid cycle . This paper reports the isolation and kinetic characterization of wild-type and five mutant PrpB proteins . Wild-type PrpB protein had a molecular mass of approximately 32 kDa per subunit, and the biologically active enzyme was comprised of four subunits . Optimal 2-MIC lyase activity was measured at pH 7.5 and 50°C, and the reaction required Mg2+ ions; equimolar concentrations of Mn2+ ions were a poor substitute for Mg2+ (28% specific activity) . Dithiothreitol (DTT) or reduced glutathione (GSH) was required for optimal activity; the role of DTT or GSH was apparently not to reduce disulfide bonds, since the disulfide-specific reducing agent Tris(2-carboxyethyl)phosphine hydrochloride failed to substitute for DTT or GSH . The Km of PrpB for 2-MIC was measured at 19 µM, with a kcat of 105 s-1 . Mutations in the prpB gene were introduced by site-directed mutagenesis based on the active-site residues deemed important for catalysis in the closely related phosphoenolpyruvate mutase and isocitrate lyase enzymes . Residues D58, K121, C123, and H125 of PrpB were changed to alanine, and residue R122 was changed to lysine . Nondenaturing polyacrylamide gel electrophoresis indicated that all mutant PrpB proteins retained the same oligomeric state of the wild-type enzyme, which is known to form tetramers . The PrpBK121A, PrpBH125A, and PrpBR122K mutant proteins formed enzymes that had 1,050-, 750-, and 2-fold decreases in kcat for 2-MIC lyase activity, respectively . The PrpBD58A and PrpBC123A proteins formed tetramers that displayed no detectable 2-MIC lyase activity indicating that both of these residues are essential for catalysis . Based on the proposed mechanism of the closely related isocitrate lyases, PrpB residue C123 is proposed to serve as the active site base, and residue D58 is critical for the coordination of a required Mg2+ ion . Effect of Intracellular pH on Rotational Speed of Bacterial Flagellar Motors. Tohru Minamino, 2003.Weak acids such as acetate and benzoate, which partially collapse the transmembrane proton gradient, not only mediate pH taxis but also impair the motility of Escherichia coli and Salmonella at an external pH of 5.5 . In this study, we examined in more detail the effect of weak acids on motility at various external pH values . A change of external pH over the range 5.0 to 7.8 hardly affected the swimming speed of E . coli cells in the absence of 34 mM potassium acetate . In contrast, the cells decreased their swimming speed significantly as external pH was shifted from pH 7.0 to 5.0 in the presence of 34 mM acetate . The total proton motive force of E . coli cells was not changed greatly by the presence of acetate . We measured the rotational rate of tethered E . coli cells as a function of external pH . Rotational speed decreased rapidly as the external pH was decreased, and at pH 5.0, the motor stopped completely . When the external pH was returned to 7.0, the motor restarted rotating at almost its original level, indicating that high intracellular proton (H+) concentration does not irreversibly abolish flagellar motor function . Both the swimming speeds and rotation rates of tethered cells of Salmonella also decreased considerably when the external pH was shifted from pH 7.0 to 5.5 in the presence of 20 mM benzoate . We propose that the increase in the intracellular proton concentration interferes with the release of protons from the torque-generating units, resulting in slowing or stopping of the motors . Role of Nonhost Environments in the Lifestyles of Salmonella and Escherichia coli. Mollie D. Winfield, 2003.
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