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Differential Regulation of Soluble and Membrane-Bound Inorganic Pyrophosphatases in the Photosynthetic Bacterium Rhodospirillum rubrum Provides Insights into Pyrophosphate-Based Stress Bioenergetics. Rosa L. López-Marqués, 2004.Soluble and membrane-bound inorganic pyrophosphatases (sPPase and H+-PPase, respectively) of the purple nonsulfur bacterium Rhodospirillum rubrum are differentially regulated by environmental growth conditions . Both proteins and their transcripts were found in cells of anaerobic phototrophic batch cultures along all growth phases, although they displayed different time patterns . However, in aerobic cells that grow in the dark, which exhibited the highest growth rates, Northern and Western blot analyses as well as activity assays demonstrated high sPPase levels but no H+-PPase . It is noteworthy that H+-PPase is highly expressed in aerobic cells under acute salt stress (1 M NaCl) . H+-PPase was also present in anaerobic cells growing at reduced rates in the dark under either fermentative or anaerobic respiratory conditions . Since H+-PPase was detected not only under all anaerobic growth conditions but also under salt stress in aerobiosis, the corresponding gene is not invariably repressed by oxygen . Primer extension analyses showed that, under all anaerobic conditions tested, the R . rubrum H+-PPase gene utilizes two activator-dependent tandem promoters, one with an FNR-like sequence motif and the other with a RegA motif, whereas in aerobiosis under salt stress, the H+-PPase gene is transcribed from two further tandem promoters involving other transcription factors . These results demonstrate a tight transcriptional regulation of the H+-PPase gene, which appears to be induced in response to a variety of environmental conditions, all of which constrain cell energetics . Staphylococcus aureus Contains Two Low-Molecular-Mass Phosphotyrosine Protein Phosphatases. Didier Soulat, 2002.The analysis of the different amino acid sequences deduced from the complete genome sequence of the gram-positive bacterium Staphylococcus aureus suggested the presence of two eukaryotic-protein-like low-molecular-mass phosphotyrosine protein phosphatases, which are usually found in gram-negative bacteria . To check this prediction, the corresponding genes were cloned and overexpressed in an Escherichia coli system . Two distinct proteins with an apparent molecular mass of 23 kDa each, PtpA and PtpB, were produced and then purified by affinity chromatography and assayed for enzymatic properties . As expected, they both exhibited phosphatase activity in vitro, with a maximum value at a pH of around 6.2 and at a temperature of 40°C . In addition, their kinetic constants, their specificity for phosphotyrosine residues, and their sensitivity to two phosphatase inhibitors, N-ethylmaleimide and orthovanadate, matched those of acid low-molecular-mass phosphotyrosine protein phosphatases . Identification of a Novel Membrane-Associated Gene Product That Suppresses Toxicity of a TrfA Peptide from Plasmid RK2 and Its Relationship to the DnaA Host Initiation Protein. Peter D. Kim, 2003.The toxicity of a peptide derived from the amino-terminal portion of 33-kDa TrfA, one of the initiation proteins encoded by the broad-host-range plasmid RK2, was suppressed by a host protein related to DnaA, the initiation protein of Escherichia coli . The newly identified 28.4-kDa protein, termed a DnaA paralog (Dp) because it is similar to a region of DnaA but likely has a different function in initiation of plasmid RK2 replication, interacts physically with the 33-kDa TrfA initiation protein, including the initiation-active monomeric form . The Dp has a cellular distribution similar to that of the 33-kDa TrfA initiation protein, being found primarily in the inner membrane fraction, with lesser amounts detected in the outer membrane fraction and almost none in the soluble fraction of E . coli . Maintenance and inner membrane-associated replication of plasmid RK2 were enhanced in a Dp knockout strain and inhibited in strains containing extra copies of the Dp gene or in membrane extracts to which a tagged form of Dp was added . Recently, the Dp was independently shown to help prevent overinitiation in E . coli and was termed Hda (S . Kato and T . Katayama, EMBO J . 20:4253-4262, 2001) .
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