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Fine-Tuning in Regulation of Clp Protein Content in Bacillus subtilis. Ulf Gerth, 2004.Clp-controlled proteolysis in Bacillus subtilis seems to play a substantial role, particularly under stress conditions . Calibrated Western blot analyses were used to estimate the approximate numbers of heat-inducible Clp molecules within a single cell . According to these numbers, the different Clp ATPases do not seem to compete for the proteolytic subunit ClpP . Coimmunoprecipitation experiments revealed the predicted specific ClpX-ClpP, ClpC-ClpP, and ClpE-ClpP interactions . ClpE and ClpX are rapidly degraded in wild-type cells during permanent heat stress but remained almost stable in a clpP mutant, suggesting ClpP-dependent degradation . In particular, ClpCP appeared to be involved in the degradation of the short-lived ClpE ATPase, indicating a negative "autoregulatory" circuit for this particular Clp ATPase at the posttranslational level . Analysis of the half-life of stress-inducible clp mRNAs during exponential growth and heat shock revealed precise regulation of the synthesis of each Clp protein at the posttranscriptional level as well to meet the needs of B . subtilis .
Increasing Secretion of a Bivalent Anti-T-Cell Immunotoxin by Pichia pastoris. Jung Hee Woo, 2004.The bivalent anti-T-cell immunotoxin A-dmDT390-bisFv(G4S) was developed for treatment of T-cell leukemia and autoimmune diseases and for tolerance induction for transplantation . This immunotoxin was produced extracellularly in toxin-sensitive Pichia pastoris JW102 (Mut+) under control of the AOX1 promoter . There were two major barriers to efficient immunotoxin production, the toxicity of the immunotoxin for P . pastoris and the limited capacity of P . pastoris to secrete the immunotoxin . The immunotoxin toxicity resulted in a decrease in the methanol consumption rate, cessation of cell growth, and low immunotoxin productivity after the first 22 h of methanol induction . Continuous cell growth and continuous immunotoxin secretion after the first 22 h of methanol induction were obtained by adding glycerol to the methanol feed by using a 4:1 methanol-glycerol mixed feed as an energy source and by continuously adding a yeast extract solution during methanol induction . The secretory capacity was increased from 22.5 to 37 mg/liter by lowering the induction temperature . A low temperature reduced the methanol consumption rate and protease activity in the supernatant but not cell growth . The effects of adding glycerol and yeast extract to the methanol feed were synergistic . Adding yeast extract primarily enhanced methanol utilization and cell growth, while adding glycerol primarily enhanced immunotoxin production . The synergy was further enhanced by decreasing the induction temperature from 23 to 15°C, which resulted in a robust process with a yield of 37 mg/liter, which was sevenfold greater than the yield previously reported for a toxin-resistant CHO cell expression system . This methodology should be applicable to other toxin-related recombinant proteins in toxin-sensitive P . pastoris . Determinants of the C-Terminal Domain of the Escherichia coli RNA Polymerase  Subunit Important for Transcription at Class I Cyclic AMP Receptor Protein-Dependent Promoters.Nigel J. Savery, 2002.Alanine scanning of the Escherichia coli RNA polymerase subunit C-terminal domain (CTD) was used to identify amino acid side chains important for class I cyclic AMP receptor protein (CRP)-dependent transcription . Key residues were investigated further in vivo and in vitro . Substitutions in three regions of
CTD affected class I CRP-dependent transcription from the CC(-61.5) promoter and/or the lacP1 promoter . These regions are (i) the 287 determinant, previously shown to contact CRP during class II CRP-dependent transcription; (ii) the 265 determinant, previously shown to be important for
CTD-DNA interactions, including those required for class II CRP-dependent transcription; and (iii) the 261 determinant . We conclude that CRP contacts the same target in
CTD, the 287 determinant, at class I and class II CRP-dependent promoters . We also conclude that the relative contributions of individual residues within the 265 determinant depend on promoter sequence, and we discuss explanations for effects of substitutions in the 261 determinant .
The Iron-Responsive Regulator Fur Is Transcriptionally Autoregulated and Not Essential in Neisseria meningitidis. Isabel Delany, 2003.Fur is a well-known iron-responsive repressor of gene transcription, which is used by many bacteria to respond to the low-iron environment that pathogens encounter during infection . The fur gene in Neisseria meningitidis has been described as an essential gene that may regulate a broad array of genes . We succeeded in obtaining an N . meningitidis mutant with the fur gene knocked out and used it to undertake studies of fur-mediated iron regulation . We show that expression of both Fur and the transferrin binding protein Tbp2 is iron regulated and demonstrate that this regulation is Fur mediated for the Tbp2 protein . Footprinting analysis revealed that Fur binds to two distinct sites upstream of its coding region with different affinities and that these binding sites overlap two promoters that differentially control transcription of the fur gene in response to iron . The presence of two independently regulated fur promoters may allow meningococcus to fine-tune expression of this regulator controlling iron homeostasis, possibly during infection .
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