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Mutations Altering the N-Terminal Receiver Domain of NRI (NtrC) That Prevent Dephosphorylation by the NRII-PII Complex in Escherichia coli. Augen A. Pioszak, 2004.The phosphorylated form of NRI is the transcriptional activator of nitrogen-regulated genes in Escherichia coli . NRI  P
displays a slow autophosphatase activity and is rapidly
dephosphorylated by the complex of the NRII and PII signal
transduction proteins . Here we describe the isolation of two
mutations, causing the alterations
 D10
and K104Q in the receiver domain of NRI, that were selected as
conferring resistance to dephosphorylation by the NRII-PII complex .
The mutations, which alter highly conserved residues near the D54
site of phosphorylation in the NRI receiver domain, resulted in
elevated expression of nitrogen-regulated genes under nitrogen-rich
conditions . The altered NRI receiver domains were phosphorylated by
NRII in vitro but were defective in dephosphorylation . The
D10
receiver domain retained normal autophosphatase activity but was
resistant to dephosphorylation by the NRII-PII complex . The K104Q
receiver domain lacked both the autophosphatase activity and the
ability to be dephosphorylated by the NRII-PII complex . The
properties of these altered proteins are consistent with the
hypothesis that the NRII-PII complex is not a true phosphatase but
rather collaborates with NRI P
to bring about its dephosphorylation .
Use of Microautoradiography Combined with Fluorescence In Situ Hybridization To Determine Dimethylsulfoniopropionate Incorporation by Marine Bacterioplankton Taxa. Maria Vila, 2004. Antigen 43-Mediated Autotransporter Display, a Versatile Bacterial Cell Surface Presentation System. Kristian Kjærgaard, 2002.Antigen 43 (Ag43), a self-recognizing outer membrane protein of Escherichia coli, has been converted into an efficient and versatile tool for surface display of foreign protein segments . Ag43 is an autotransporter protein characterized by the feature that all information required for transport to the outer membrane and secretion through the cell envelope is contained within the protein itself . Ag43 consists of two subunits (  and ß), where the ß-subunit forms an integral outer membrane translocator to which the
-subunit is noncovalently attached . The simplicity of the Ag43 system makes it ideally suited as a surface display scaffold . Here we demonstrate that the Ag43
-module can accommodate and display correctly folded inserts and has the ability to display entire functional protein domains, exemplified by the FimH lectin domain . The presence of heterologous cysteine bridges does not interfere with surface display, and Ag43 chimeras are correctly processed into
- and ß-modules, offering optional and easy release of the chimeric
-subunits . Furthermore, Ag43 can be displayed in many gram-negative bacteria . This feature is exploited for display of our chimeras in an attenuated Salmonella strain .
Novel Medium-Chain Prenyl Diphosphate Synthase from the Thermoacidophilic Archaeon Sulfolobus solfataricus. Hisashi Hemmi, 2002.Two open reading frames which encode the homologues of (all-E) prenyl diphosphate synthase are found in the whole-genome sequence of Sulfolobus solfataricus, a thermoacidophilic archaeon . It has been suggested that one is a geranylgeranyl diphosphate synthase gene, but the specificity and biological significance of the enzyme encoded by the other have remained unclear . Thus, we isolated the latter by the PCR method, expressed the enzyme in Escherichia coli cells, purified it, and characterized it . The archaeal enzyme, 281 amino acids long, is highly thermostable and requires Mg2+ and Triton X-100 for full activity . It catalyzes consecutive E-type condensations of isopentenyl diphosphate with an allylic substrate such as geranylgeranyl diphosphate and yields the medium-chain product hexaprenyl diphosphate . Despite such product specificity, phylogenetic analysis revealed that the archaeal medium-chain prenyl diphosphate synthase is distantly related to the other medium- and long-chain enzymes but is closely related to eucaryal short-chain enzymes . IS981-Mediated Adaptive Evolution Recovers Lactate Production by ldhB Transcription Activation in a Lactate Dehydrogenase-Deficient Strain of Lactococcus lactis. Roger S. Bongers, 2003.Lactococcus lactis NZ9010 in which the las operon-encoded ldh gene was replaced with an erythromycin resistance gene cassette displayed a stable phenotype when grown under aerobic conditions, and its main end products of fermentation under these conditions were acetate and acetoin . However, under anaerobic conditions, the growth of these cells was strongly retarded while the main end products of fermentation were acetate and ethanol . Upon prolonged subculturing of this strain under anaerobic conditions, both the growth rate and the ability to produce lactate were recovered after a variable number of generations . This recovery was shown to be due to the transcriptional activation of a silent ldhB gene coding for an Ldh protein (LdhB) with kinetic parameters different from those of the native las operon-encoded Ldh protein . Nevertheless, cells producing LdhB produced mainly lactate as the end product of fermentation . The mechanism underlying the ldhB gene activation was primarily studied in a single-colony isolate of the recovered culture, designated L . lactis NZ9015 . Integration of IS981 in the upstream region of ldhB was responsible for transcription activation of the ldhB gene by generating an IS981-derived -35 promoter region at the correct spacing with a natively present -10 region . Subsequently, analysis of 10 independently isolated lactate-producing derivatives of L . lactis NZ9010 confirmed that the ldhB gene is transcribed in all of them . Moreover, characterization of the upstream region of the ldhB gene in these derivatives indicated that site-specific and directional IS981 insertion represents the predominant mechanism of the observed recovery of the ability to produce lactate . Microbial Conversion of Corn Stalks to Riches. Roy H. Doi, 2003. Metabolic Engineering of Escherichia coli for Production of Enantiomerically Pure (R)-(-)-Hydroxycarboxylic Acids. Sang Yup Lee, 2003.A heterologous metabolism of polyhydroxyalkanoate (PHA) biosynthesis and degradation was established in Escherichia coli by introducing the Ralstonia eutropha PHA biosynthesis operon along with the R . eutropha intracellular PHA depolymerase gene . By with this metabolically engineered E . coli, enantiomerically pure (R)-3-hydroxybutyric acid (R3HB) could be efficiently produced from glucose . By employing a two-plasmid system, developed as the PHA biosynthesis operon on a medium-copy-number plasmid and the PHA depolymerase gene on a high-copy-number plasmid, R3HB could be produced with a yield of 49.5% (85.6% of the maximum theoretical yield) from glucose . By integration of the PHA biosynthesis genes into the chromosome of E . coli and by introducing a plasmid containing the PHA depolymerase gene, R3HB could be produced without plasmid instability in the absence of antibiotics . This strategy can be used for the production of various enantiomerically pure (R)-hydroxycarboxylic acids from renewable resources .
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