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Phage Shock Protein PspA of Escherichia coli Relieves Saturation of Protein Export via the Tat Pathway. Matthew P. DeLisa, 2004.Overexpression of either heterologous or homologous proteins that are routed to the periplasm via the twin-arginine translocation (Tat) pathway results in a block of export and concomitant accumulation of the respective protein precursor in the cytoplasm . Screening of a plasmid-encoded genomic library for mutants that confer enhanced export of a TorA signal sequence (ssTorA)-GFP-SsrA fusion protein, and thus result in higher cell fluorescence, yielded the pspA gene encoding phage shock protein A . Coexpression of pspA relieved the secretion block observed with ssTorA-GFP-SsrA or upon overexpression of the native Tat proteins SufI and CueO . A similar effect was observed with the Synechocystis sp . strain PCC6803 PspA homologue, VIPP1, indicating that the role of PspA in Tat export may be phylogenetically conserved . Mutations in Tat components that completely abolish export result in a marked induction of PspA protein synthesis, consistent with its proposed role in enhancing protein translocation via Tat . Forespore Signaling Is Necessary for Pro-  K Processing during Bacillus subtilis Sporulation Despite the Loss of SpoIVFA upon Translational Arrest.Lee Kroos, 2002.The K checkpoint coordinates gene expression in the mother cell with signaling from the forespore during Bacillus subtilis sporulation . The signaling pathway involves SpoIVB, a serine peptidase produced in the forespore, which is believed to cross the innermost membrane surrounding the forespore and activate a complex of proteins, including BofA, SpoIVFA, and SpoIVFB, located in the outermost membrane surrounding the forespore . Activation of the complex allows proteolytic processing of pro-K, and the resulting
K RNA polymerase transcribes genes in the mother cell . To investigate activation of the pro-K processing complex, the level of SpoIVFA in extracts of sporulating cells was examined by Western blot analysis . The SpoIVFA level decreased when pro-K processing began during sporulation . In extracts of a spoIVB mutant defective in forespore signaling, the SpoIVFA level failed to decrease normally and no processing of pro-K was observed . Although these results are consistent with a model in which SpoIVFA inhibits processing until the SpoIVB-mediated signal is received from the forespore, we discovered that loss of SpoIVFA was insufficient to allow processing under certain conditions, including static incubation of the culture and continued shaking after the addition of inhibitors of oxidative phosphorylation or translation . Under these conditions, loss of SpoIVFA was independent of spoIVB . The inability to process pro-K under these conditions was not due to loss of SpoIVFB, the putative processing enzyme, or to a requirement for ongoing synthesis of pro-K . Rather, it was found that the requirements for shaking of the culture, for oxidative phosphorylation, and for translation could be bypassed by mutations that uncouple processing from dependence on forespore signaling . This suggests that ongoing translation is normally required for efficient pro-K processing because synthesis of the SpoIVB signal protein is needed to activate the processing complex . When translation is blocked, synthesis of SpoIVB ceases, and the processing complex remains inactive despite the loss of SpoIVFA . Taken together, the results suggest that SpoIVB signaling activates the processing complex by performing another function in addition to causing loss of SpoIVFA or by causing loss of SpoIVFA in a different way than when translation is blocked . The results also demonstrate that the processing machinery can function in the absence of translation or an electrochemical gradient across membranes .
Role of 2-Phosphoglycolate Phosphatase of Escherichia coli in Metabolism of the 2-Phosphoglycolate Formed in DNA Repair. Maria Teresa Pellicer, 2003.The enzyme 2-phosphoglycolate phosphatase from Escherichia coli, encoded by the gph gene, was purified and characterized . The enzyme was highly specific for 2-phosphoglycolate and showed good catalytic efficiency (kcat/Km), which enabled the conversion of this substrate even at low intracellular concentrations . A comparison of the structural and functional features of this enzyme with those of 2-phosphoglycolate phosphatases of different origins showed a high similarity of the sequences, implying the use of the same catalytic mechanism . Western blot analysis revealed constitutive expression of the gph gene, regardless of the carbon source used, growth stage, or oxidative stress conditions . We showed that this housekeeping enzyme is involved in the dissimilation of the intracellular 2-phosphoglycolate formed in the DNA repair of 3'-phosphoglycolate ends . DNA strand breaks of this kind are caused by agents such as the radiomimetic compound bleomycin . The differential response between a 2-phosphoglycolate phosphatase-deficient mutant and its parental strain after treatment with bleomycin allowed us to connect the intracellular formation of 2-phosphoglycolate with the production of glycolate, which is subsequently incorporated into general metabolism . We thus provide evidence for a salvage function of 2-phosphoglycolate phosphatase in the metabolism of a two-carbon compound generated by the cellular DNA repair machinery . Electricity Production by Geobacter sulfurreducens Attached to Electrodes. Daniel R. Bond, 2003.Previous studies have suggested that members of the Geobacteraceae can use electrodes as electron acceptors for anaerobic respiration . In order to better understand this electron transfer process for energy production, Geobacter sulfurreducens was inoculated into chambers in which a graphite electrode served as the sole electron acceptor and acetate or hydrogen was the electron donor . The electron-accepting electrodes were maintained at oxidizing potentials by connecting them to similar electrodes in oxygenated medium (fuel cells) or to potentiostats that poised electrodes at +0.2 V versus an Ag/AgCl reference electrode (poised potential) . When a small inoculum of G . sulfurreducens was introduced into electrode-containing chambers, electrical current production was dependent upon oxidation of acetate to carbon dioxide and increased exponentially, indicating for the first time that electrode reduction supported the growth of this organism . When the medium was replaced with an anaerobic buffer lacking nutrients required for growth, acetate-dependent electrical current production was unaffected and cells attached to these electrodes continued to generate electrical current for weeks . This represents the first report of microbial electricity production solely by cells attached to an electrode . Electrode-attached cells completely oxidized acetate to levels below detection (<10 µM), and hydrogen was metabolized to a threshold of 3 Pa . The rates of electron transfer to electrodes (0.21 to 1.2 µmol of electrons/mg of protein/min) were similar to those observed for respiration with Fe(III) citrate as the electron acceptor (Eo' =+0.37 V) . The production of current in microbial fuel cell (65 mA/m2 of electrode surface) or poised-potential (163 to 1,143 mA/m2) mode was greater than what has been reported for other microbial systems, even those that employed higher cell densities and electron-shuttling compounds . Since acetate was completely oxidized, the efficiency of conversion of organic electron donor to electricity was significantly higher than in previously described microbial fuel cells . These results suggest that the effectiveness of microbial fuel cells can be increased with organisms such as G . sulfurreducens that can attach to electrodes and remain viable for long periods of time while completely oxidizing organic substrates with quantitative transfer of electrons to an electrode .
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