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Critical Regions of secM That Control Its Translation and Secretion and Promote Secretion-Specific secA Regulation. Shameema Sarker, 2002.SecA is an essential ATP-driven motor protein that binds to presecretory or membrane proteins and the translocon and promotes the translocation or membrane integration of these proteins . secA is subject to a protein secretion-specific form of regulation, whereby its translation is elevated during secretion-limiting conditions . A novel mechanism that promotes this regulation involves translational pausing within the gene upstream of secA, secM . The secM translational pause prevents formation of an RNA helix that normally blocks secA translational initiation . The duration of this pause is controlled by the rate of secretion of nascent SecM, which in turn depends on its signal peptide and a functional translocon . We characterized the atypical secM signal peptide and found that mutations within the amino-terminal region specifically affect the secM translational pause and secA regulation, while mutations in the hydrophobic core region affect SecM secretion as well as translational pausing and secA regulation . In addition, mutational analysis of the 3' end of secM allowed us to identify a conserved region that is required to promote the translational pause that appears to be operative at the peptide level . Together, our results provide direct support for the secM translational pause model of secA regulation, and they pinpoint key sequences within secM that promote this important regulatory system . Structure-Function Studies of Escherichia coli RpoH (  32) by In Vitro Linker Insertion Mutagenesis.Franz Narberhaus, 2003.The sigma factor RpoH ( 32) is the key regulator of the heat shock response in Escherichia coli . Many structural and functional properties of the sigma factor are poorly understood . To gain further insight into RpoH regions that are either important or dispensable for its cellular activity, we generated a collection of tetrapeptide insertion variants by a recently established in vitro linker insertion mutagenesis technique . Thirty-one distinct insertions were obtained, and their sigma factor activity was analyzed by using a groE-lacZ reporter fusion in an rpoH-negative background . Our study provides a map of permissive sites which tolerate linker insertions and of functionally important regions at which a linker insertion impairs sigma factor activity . Selected linker insertion mutants will be discussed in the light of known sigma factor properties and in relation to a modeled structure of an RpoH fragment containing region 2 .
Effect of Temperature, pH, and Initial Cell Number on luxCDABE and nah Gene Expression during Naphthalene and Salicylate Catabolism in the Bioreporter Organism Pseudomonas putida RB1353. Jonathan G. Dorn, 2003.One limitation of employing lux bioreporters to monitor in situ microbial gene expression in dynamic, laboratory-scale systems is the confounding variability in the luminescent responses . For example, despite careful control of oxygen tension, growth stage, and cell number, luminescence from Pseudomonas putida RB1353, a naphthalene-degrading lux bioreporter, varied by more than sevenfold during saturated flow column experiments in our laboratory . Therefore, this study was conducted to determine what additional factors influence the luminescent response . Specifically, this study investigated the impact of temperature, pH, and initial cell number (variations within an order of magnitude) on the peak luminescence of P . putida RB1353 and the maximum degradation rate (Vmax) during salicylate and naphthalene catabolism . Statistical analyses based on general linear models indicated that under constant oxygen tension, temperature and pH accounted for 98.1% of the variability in luminescence during salicylate catabolism and 94.2 and 49.5% of the variability in Vmax during salicylate and naphthalene catabolism, respectively . Temperature, pH, and initial substrate concentration accounted for 99.9% of the variability in luminescence during naphthalene catabolism . Initial cell number, within an order of magnitude, did not have a significant influence on either peak luminescence or Vmax during salicylate and naphthalene catabolism . Over the ranges of temperature and pH evaluated, peak luminescence varied by more than 4 orders of magnitude . The minimum parameter deviation required to alter lux gene expression during salicylate and naphthalene catabolism was a change in temperature of 1°C, a change in pH of 0.2, or a change in initial cell number of 1 order of magnitude . Results from this study indicate that there is a need for careful characterization of the impact of environmental conditions on both the expression of the reporter and catabolic genes and the activities of the gene products . For example, even though lux gene expression was occurring at  35°C, the luciferase enzyme was inactive . Furthermore, this study demonstrates that with careful characterization and standardization of measurement conditions, the attainment of a reproducible luminescent response and an understanding of the response are feasible .
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