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Arginine Biosynthesis in Thermotoga maritima: Characterization of the Arginine-Sensitive N-Acetyl-L-Glutamate Kinase. M. Leonor Fernández-Murga, 2004.To help clarify the control of arginine synthesis in Thermotoga maritima, the putative gene [argB] for N-acetyl-L-glutamate kinase [NAGK] from this microorganism was cloned and overexpressed, and the resulting protein was purified and shown to be a highly thermostable and specific NAGK that is potently and selectively inhibited by arginine . Therefore, NAGK is in T . maritima the feedback control point of arginine synthesis, a process thatin this organism involves acetyl group recycling and appearsnot to involve classical acetylglutamate synthase . The inhibitionof NAGK by arginine was found to be pH independent and to dependsigmoidally on the concentration of arginine, with a Hill coefficient[N] of  4,
and the 50% inhibitory arginine concentration [I0.5]was
shown to increase with temperature, approaching above 65°Cthe I0.50
observed at 37°C with the mesophilic NAGK of Pseudomonas
aeruginosa [the best-studied arginine-inhibitable NAGK] . At75°C,
the inhibition by arginine of T . maritima NAGK wasdue to a
large increase in the Km for acetylglutamate triggered
by the inhibitor, but at 37°C arginine also substantially
decreased the Vmax of the enzyme . The NAGKs of T . maritima
andP . aeruginosa behaved in gel filtration as hexamers,
justifyingthe sigmoidicity and high Hill coefficient of arginine
inhibition,and arginine or the substrates failed to disaggregate
theseenzymes . In contrast, Escherichia coli NAGK is not
inhibitedby arginine and is dimeric, and thus the hexameric
architecturemay be an important determinant of arginine sensitivity .
Potentialthermostability determinants of T . maritima NAGK are
also discussed.
Requirement of the Listeria monocytogenes Broad-Range Phospholipase PC-PLC during Infection of Human Epithelial Cells. Angelika Gründling, 2003.In this study, we investigated the requirement of the Listeria monocytogenes broad-range phospholipase C (PC-PLC) during infection of human epithelial cells . L . monocytogenes is a facultative intracellular bacterial pathogen of humans and a variety of animal species . After entering a host cell, L . monocytogenes is initially surrounded by a membrane-bound vacuole . Bacteria promote their escape from this vacuole, grow within the host cell cytosol, and spread from cell to cell via actin-based motility . Most infection studies with L . monocytogenes have been performed with mouse cells or an in vivo mouse model of infection . In all mouse-derived cells tested, the pore-forming cytolysin listeriolysin O (LLO) is absolutely required for lysis of primary vacuoles formed during host cell entry . However, L . monocytogenes can escape from primary vacuoles in the absence of LLO during infection of human epithelial cell lines Henle 407, HEp-2, and HeLa . Previous studies have shown that the broad-range phospholipase C, PC-PLC, promotes lysis of Henle 407 cell primary vacuoles in the absence of LLO . Here, we have shown that PC-PLC is also required for lysis of HEp-2 and HeLa cell primary vacuoles in the absence of LLO expression . Furthermore, our results indicated that the amount of PC-PLC activity is critical for the efficiency of vacuolar lysis . In an LLO-negative derivative of L . monocytogenes strain 10403S, expression of PC-PLC has to increase before or upon entry into human epithelial cells, compared to expression in broth culture, to allow bacterial escape from primary vacuoles . Using a system for inducible PC-PLC expression in L . monocytogenes, we provide evidence that phospholipase activity can be increased by elevated expression of PC-PLC or Mpl, the enzyme required for proteolytic activation of PC-PLC . Lastly, by using the inducible PC-PLC expression system, we demonstrate that, in the absence of LLO, PC-PLC activity is not only required for lysis of primary vacuoles in human epithelial cells but is also necessary for efficient cell-to-cell spread . We speculate that the additional requirement for PC-PLC activity is for lysis of secondary double-membrane vacuoles formed during cell-to-cell spread . Species-Wide Variation in the Escherichia coli Flagellin (H-Antigen) Gene. Lei Wang, 2003.Escherichia coli is a clonal species . The best-understood components of its clonal variation are the flagellar (H) and polysaccharide (O) antigens, both well documented since the mid-1930s because of their use in serotyping . Flagellin is the protein subunit of the flagellum that carries H-antigen specificity . We show that 43 of the 54 H-antigen specificities of E . coli map to the flagellin gene at fliC and sequenced all 43 forms and confirmed specificity of each by cloning and expression . This is, to our knowledge, the first time that all known forms of such a highly polymorphic gene have been fully sequenced and characterized for any species . The established distinction between a highly variable central region and more conserved flanking regions is upheld . The sequences fall into two groups, one of which may be derived from the fliC gene of the E . coli/Salmonella enterica common ancestor, the other perhaps obtained by lateral transfer since species divergence . Comparison of sequences revealed that both horizontal DNA transfer and fixation of mutations under diversifying selection pressure contributed to polymorphism in this locus . A New Rate Law Describing Microbial Respiration. Qusheng Jin, 2003.The rate of microbial respiration can be described by a rate law that gives the respiration rate as the product of a rate constant, biomass concentration, and three terms: one describing the kinetics of the electron-donating reaction, one for the kinetics of the electron-accepting reaction, and a thermodynamic term accounting for the energy available in the microbe's environment . The rate law, derived on the basis of chemiosmotic theory and nonlinear thermodynamics, is unique in that it accounts for both forward and reverse fluxes through the electron transport chain . Our analysis demonstrates how a microbe's respiration rate depends on the thermodynamic driving force, i.e., the net difference between the energy available from the environment and energy conserved as ATP . The rate laws commonly applied in microbiology, such as the Monod equation, are specific simplifications of the general law presented . The new rate law is significant because it affords the possibility of extrapolating in a rigorous manner from laboratory experiment to a broad range of natural conditions, including microbial growth where only limited energy is available . The rate law also provides a new explanation of threshold phenomena, which may reflect a thermodynamic equilibrium where the energy released by electron transfer balances that conserved by ADP phosphorylation .
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