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Analysis of Infection Thread Development Using Gfp- and DsRed-Expressing Sinorhizobium meliloti. Daniel J. Gage, 2002. Purification and Biochemical Characterization of the F1Fo-ATP Synthase from Thermoalkaliphilic Bacillus sp . Strain TA2.A1. Gregory M. Cook, 2003.We describe here purification and biochemical characterization of the F1Fo-ATP synthase from the thermoalkaliphilic organism Bacillus sp . strain TA2.A1 . The purified enzyme produced the typical subunit pattern of an F1Fo-ATP synthase on a sodium dodecyl sulfate-polyacrylamide gel, with F1 subunits  , ß,
 ,
 , and
 and Fo subunits a, b, and c . The subunits were identified by N-terminal protein sequencing and mass spectroscopy . A notable feature of the ATP synthase from strain TA2.A1 was its specific blockage in ATP hydrolysis activity . ATPase activity was unmasked by using the detergent lauryldimethylamine oxide (LDAO), which activated ATP hydrolysis >15-fold . This activation was the same for either the F1Fo holoenzyme or the isolated F1 moiety, and therefore latent ATP hydrolysis activity is an intrinsic property of F1 . After reconstitution into proteoliposomes, the enzyme catalyzed ATP synthesis driven by an artificially induced transmembrane electrical potential (  ) . A transmembrane proton gradient or sodium ion gradient in the absence of
was not sufficient to drive ATP synthesis . ATP synthesis was eliminated by the electrogenic protonophore carbonyl cyanide m-chlorophenylhydrazone, while the electroneutral Na+/H+ antiporter monensin had no effect . Neither ATP synthesis nor ATP hydrolysis was stimulated by Na+ ions, suggesting that protons are the coupling ions of the ATP synthase from strain TA2.A1, as documented previously for mesophilic alkaliphilic Bacillus species . The ATP synthase was specifically modified at its c subunits by N,N'-dicyclohexylcarbodiimide, and this modification inhibited ATP synthesis .
Development of a Thermally Regulated Broad-Spectrum Promoter System for Use in Pathogenic Gram-Positive Species. David A. Schofield, 2003.Selectively regulating gene expression is an essential molecular tool that is lacking for many pathogenic gram-positive bacteria . In this report, we describe the evaluation of a series of promoters regulated by the bacteriophage P1 temperature-sensitive C1 repressor in Enterococcus faecium, Enterococcus faecalis, and Staphylococcus aureus . Using the lacZ gene to monitor gene expression, we examined the strength, basal expression, and induced expression of synthetic promoters carrying C1 operator sites . The promoters exhibited extremely low basal expression and, under inducing conditions, gave high levels of expression (100- to 1,000-fold induction) . We demonstrate that the promoter system could be modulated by temperature and showed rapid induction and that the mechanism of regulation occurred at the level of transcription . Controlled expression with the same constructs was also demonstrated in the gram-negative bacterium Escherichia coli . However, low basal expression and the ability to achieve derepression were dependent on both the number of mismatches in the C1 operator sites and the promoter driving c1 expression . Since the promoters were designed to contain conserved promoter elements from gram-positive species and were constructed in a broad-host-range plasmid, this system will provide a new opportunity for controlled gene expression in a variety of gram-positive bacteria .
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